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1.
Food Chem ; 339: 127955, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32919344

RESUMO

Lateral flow assay (LFA), performed with simple devices and short detection time, is popular in field applications. Herein, a novel sandwich type-based LFA was constructed for high sensitivity and selectivity detection of Staphylococcus aureus (S. aureus). Vancomycin-immobilized gold nanoparticles (VAN-Au NPs) were utilized as the first identifier to capture S. aureus and the specificity was guaranteed by the second recognition agent of pig immunoglobulin G (IgG). In addition, gold growth was adopted for signal amplification to further improve the detection sensitivity. S. aureus could be directly assayed by this LFA within the concentration range of 1.0 × 103-1.0 × 108 cfu mL-1 with a detection limit of 1.0 × 103 cfu mL-1. Furthermore, the novel sandwich LFA realized S. aureus detection in food samples with admissible recoveries and established a rapid, simple, cost-effective and sensitive platform, could meet the demand for on-site testing of S. aureus.


Assuntos
Antibacterianos/química , Imunoensaio/métodos , Imunoglobulina G/química , Staphylococcus aureus/isolamento & purificação , Animais , Microbiologia de Alimentos , Sucos de Frutas e Vegetais/microbiologia , Ouro/química , Imunoglobulina G/imunologia , Limite de Detecção , Nanopartículas Metálicas/química , Sistemas Automatizados de Assistência Junto ao Leito , Staphylococcus aureus/imunologia , Suínos , Vancomicina/química , Verduras/microbiologia
2.
Int J Nanomedicine ; 15: 10321-10330, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33364759

RESUMO

Background: Vaccination provides a viable alternative to antibiotics for the treatment of drug-resistant bacterial infection. Bacterial protoplasts have gained much attention for a new generation vaccine due to depleting toxic outer wall components. Purpose: The objective of this study was to reveal the effects of bacterial protoplast-derived nanovesicles (PDNVs) size on antibacterial immunity. Methods: Herein, we prepared bacterial PDNVs with different sizes by removing the cell wall of Staphylococcus aureus (S. aureus) to generate multi-antigen nanovaccines. Furthermore, we investigated the ability of PDNVs in different sizes to activate dendritic cells (DCs) and trigger humoral and cellular immune responses in vivo. Results: We obtained particles of ∼200 nm, 400 nm, and 700 nm diameters and found that all the PDNVs readily induce efficient maturation of DCs in the draining lymph nodes of the vaccinated mice. Dramatically, the activation of DCs was increased with decreasing particle sizes. In addition, vaccination with PDNVs generated elevated expression levels of specific antibody and the production of INF-γ, especially the smaller ones, indicating the capability of inducing strong humoral immunity and Th1 biased cell responses against the source bacteria. Conclusion: These observed results provide evidence for size-dependent orchestration of immune responses of PDNVs and help to rationally design and develop effective antibacterial vaccines.


Assuntos
Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Protoplastos/química , Staphylococcus aureus/citologia , Staphylococcus aureus/imunologia , Animais , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Camundongos , Nanoestruturas/química
3.
Mol Immunol ; 126: 14-24, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32739720

RESUMO

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.


Assuntos
Proteínas de Artrópodes/metabolismo , Palaemonidae/fisiologia , Proteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Mucosa Gástrica/metabolismo , Técnicas de Silenciamento de Genes , Brânquias/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Muda/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Palaemonidae/microbiologia , Proteínas/genética , Receptores Acoplados a Proteínas-G/genética , Staphylococcus aureus/imunologia , Fatores de Transcrição , Regulação para Cima/imunologia , Vibrio parahaemolyticus/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia
4.
Mol Immunol ; 126: 73-86, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32771671

RESUMO

Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 µM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Imunidade Inata , Peroxirredoxinas/imunologia , Aeromonas hydrophila/imunologia , Animais , Arguloida/imunologia , Carpas/genética , Carpas/microbiologia , Carpas/parasitologia , Clonagem Molecular , Doenças dos Peixes/sangue , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Rim Cefálico/enzimologia , Rim Cefálico/imunologia , Fígado/enzimologia , Fígado/imunologia , Estresse Oxidativo/imunologia , Peroxirredoxinas/genética , Filogenia , Poli I-C/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/imunologia
5.
Proc Natl Acad Sci U S A ; 117(37): 22992-23000, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32855300

RESUMO

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including Staphylococcus aureus, secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of S. aureus have failed to meet clinical endpoints. It is unclear that such failures can be solely attributed to the poor selection of antibody targets. Thus far, studies to delineate antibody-mediated uptake and killing of Gram-positive pathogens remain extremely limited. Here, we exploit 3F6-hIgG1, a human monoclonal antibody that binds and neutralizes the abundant surface-exposed Staphylococcal protein A (SpA). We find that galactosylation of 3F6-hIgG1 that favors C1q recruitment is indispensable for opsonophagocytic killing of staphylococci and for protection against bloodstream infection in animals. However, the simple removal of fucosyl residues, which results in reduced C1q binding and increased engagement with FcγR, maintains the opsonophagocytic killing and protective attributes of the antibody. We confirm these results by engineering 3F6-hIgG1 variants with biased binding toward C1q or FcγRs. While the therapeutic benefit of monoclonal antibodies against infectious disease agents may be debatable, the functional characterization of such antibodies represents a powerful tool for the development of correlates of protection that may guide future vaccine trials.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Fagocitose/imunologia , Proteína Estafilocócica A/imunologia , Animais , Linhagem Celular , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia
6.
PLoS Pathog ; 16(8): e1008733, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817694

RESUMO

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.


Assuntos
Saccharomyces cerevisiae/imunologia , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Coagulase/administração & dosagem , Coagulase/genética , Coagulase/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae/química , Infecções Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Staphylococcus aureus/genética , Células Th1/imunologia , Células Th17/imunologia , Vacinação , beta-Glucanas/administração & dosagem , beta-Glucanas/imunologia
7.
Mol Immunol ; 126: 1-7, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32712503

RESUMO

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/metabolismo , Imunidade Inata/genética , Palaemonidae/imunologia , Fatores de Transcrição/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Aquicultura , Proteínas de Artrópodes/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Palaemonidae/genética , Palaemonidae/metabolismo , Palaemonidae/microbiologia , Poli I-C/imunologia , Staphylococcus aureus/imunologia , Fatores de Transcrição/genética , Vibrio parahaemolyticus/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-32637365

RESUMO

The airway epithelial barrier is a major barrier protecting against clinically significant infections of the lung. Its integrity is often compromised due to mechanical, chemical, or infectious causes. Opportunistic bacterial pathogens are poised to cause parenchymal infection and become difficult to eradicate due to adaptive metabolic changes, biofilm formation, and the acquisition of antimicrobial resistance and fitness genes. Enhancing mucosal defenses by modulating the cytokines that regulate barrier functions, such as interleukin-22 (IL-22) and interferon-λ (IFN-λ), members of the IL-10 family of cytokines, is an attractive approach to prevent these infections that are associated with high morbidity and mortality. These cytokines both signal through the cognate receptor IL-10RB, have related protein structures and common downstream signaling suggesting shared roles in host respiratory defense. They are typically co-expressed in multiple models of infections, but with differing kinetics. IL-22 has an important role in the producing antimicrobial peptides, upregulating expression of junctional proteins in the airway epithelium and working in concert with other inflammatory cytokines such as IL-17. Conversely, IFN-λ, a potent antiviral in influenza infection with pro-inflammatory properties, appears to decrease junctional integrity allowing for bacterial and immune cell translocation. The effects of these cytokines are pleotropic, with pathogen and tissue specific consequences. Understanding how these cytokines work in the mucosal defenses of the respiratory system may suggest potential targets to prevent invasive infections of the damaged lung.


Assuntos
Interferon gama/imunologia , Subunidade beta de Receptor de Interleucina-10/imunologia , Interleucinas/imunologia , Mucosa Respiratória/imunologia , Junções Íntimas/imunologia , Infecções por Coronavirus/imunologia , Humanos , Influenza Humana/imunologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/microbiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia
9.
Mol Immunol ; 124: 211-217, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32603960

RESUMO

The primary immune response against Staphylococcus aureus is mediated by neutrophils. In response to S. aureus and its proteins, neutrophil shows two different kinds of NETosis, viz. suicidal and vesicular NETosis. Glucose is the major energy source of neutrophils for performing NETosis. However, NETosis was found altered in response to high glucose levels. Growth of S. aureus was also found modulated in response to high glucose and they behave differently at different glucose levels. This work was attempted to study NET release in response to S. aureus cell-free culture supernatant at different glucose concentrations. Freshly isolated neutrophils were treated with different concentrations of glucose along with S. aureus cell-free culture supernatant and were analyzed for neutrophil extracellular trap formation, ROS production, and peptidylarginine deiminase 4 activities. Influence of calcium on NETosis was analyzed using calcium chelator (EDTA) and calcium inhibitor (TMB-8). With increasing glucose levels, NET release in response to S. aureus cell-free culture supernatant was increased. Oxidant level was also increased dose-dependently with increasing concentrations of glucose. At very high glucose concentrations (> 15 mM), vesicular NETosis was predominantly observed. At these glucose concentrations, peptidylarginine deiminase activity was found to be decreased. Furthermore, calcium quenching in the medium facilitated vesicular mode of NET release. In conclusion, calcium depletion occurring at high glucose concentrations can reduce peptidylarginine deiminase 4 activity and can thereby promote the vesicular NET release.


Assuntos
Cálcio/metabolismo , Armadilhas Extracelulares/metabolismo , Glucose/metabolismo , Neutrófilos/imunologia , Staphylococcus aureus/imunologia , Células Cultivadas , Armadilhas Extracelulares/imunologia , Humanos , Vesículas Secretórias/metabolismo , Infecções Estafilocócicas/imunologia
10.
Nat Commun ; 11(1): 3334, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620760

RESUMO

TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-ß signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes.


Assuntos
Homeostase/imunologia , Inflamação/imunologia , Interleucina-10/imunologia , Intestinos/imunologia , Células Th17/imunologia , Animais , Plasticidade Celular/imunologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Células HEK293 , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Células Th17/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo
11.
PLoS One ; 15(4): e0225873, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352968

RESUMO

Black soldier fly (BSF; Hermetia illucens L.) larvae can convert fresh pig manure into protein and fat-rich biomass, which can then be used as aquafeed for select species. Currently, BSF is the only approved insect for such purposes in Canada, USA, and the European Union. Pig manure could serve as a feed substrate for BSF; however, it is contaminated with zoonotic pathogens (e.g., Staphylococcus aureus and Salmonella spp.). Fortunately, BSF larvae inhibit many of these zoonotic pathogens; however, the mechanisms employed are unclear. We employed RNAi, qRT-PCR, and Illumina MiSeq 16S rDNA high-throughput sequencing to examine the interaction between two immune genes (Duox in Duox-reactive oxygen species [ROS] immune system and TLR3 in the Toll signaling pathway) and select pathogens common in pig manure to decipher the mechanisms resulting in pathogen suppression. Results indicate Bsf Duox-TLR3 RNAi increased bacterial load but decreased relative abundance of Providencia and Dysgonomonas, which are thought to be commensals in the BSF larval gut. Bsf Duox-TLR3 RNAi also inactivated the NF-κB signaling pathway, downregulated the expression of antimicrobial peptides, and diminished inhibitory effects on zoonotic pathogen. The resulting dysbiosis stimulated an immune response by activating BsfDuox and promoting ROS, which regulated the composition and structure of the gut bacterial community. Thus, BsfDuox and BsfTLR3 are important factors in regulating these key gut microbes, while inhibiting target zoonotic pathogens.


Assuntos
Oxidases Duais/imunologia , Microbioma Gastrointestinal , Proteínas de Insetos/imunologia , Esterco/microbiologia , Simuliidae/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Homeostase , Humanos , Larva/imunologia , Larva/microbiologia , Salmonella/imunologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Transdução de Sinais , Simuliidae/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Suínos , Zoonoses/imunologia , Zoonoses/microbiologia
12.
PLoS Pathog ; 16(5): e1008393, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433711

RESUMO

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.


Assuntos
Memória Imunológica/imunologia , Vírus da Influenza A/imunologia , Superantígenos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Feminino , Humanos , Memória Imunológica/fisiologia , Vírus da Influenza A/metabolismo , Influenza Humana/imunologia , Influenza Humana/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Staphylococcus aureus/imunologia , Superantígenos/fisiologia , Superinfecção/imunologia , Vacinação
13.
Mikrochim Acta ; 187(5): 290, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32342176

RESUMO

A surface-enhanced Raman scattering (SERS)-based immunocapture nanoprobe is described for the detection of pathogenic bacteria. The probe uses boronic acid-functionalized polydopamine-coated Au@Ag nanoparticles as an advanced SERS nanotag. Modified magnetic IgG@Fe3O4 nanoparticles are used for magnetic separation. Au@Ag@PDA nanoparticles, where PDA stands for polydopamine, were functionalized with boronic acid to bind to pathogenic bacteria and induce signal amplification. The Raman signal is amplified 108 times when the SERS tag binds the surface of bacteria. The SERS spectra exhibit fingerprint-like patterns that enable bacterial classification. The results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the spectral regions were compared. The bacterial surface protein and glycan signals (1300-1450 cm-1) were the best regions for bacterial classification. Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, and Klebsiella pneumonia were successfully classified by this method. The lowest detection limit was 10 colonies/mL (CFU·mL). The assay can be completed within 30 min. Conceivably, this method may be extended to the quantitative detection or classification of bacteria under various other conditions. Graphical abstract Schematic representation of immunocapture and detection of pathogenic bacteria using boronic acid-functionalized polydopamine-coated Au@Ag nanoprobe through the bacterial surface protein and glycan signals. Green arrow: laser; black arrow: SERS; red ball: bacteria; grey ball: IgG@Fe3O4; golden ball: boronic acid-functionalized Au@Ag@PDA.


Assuntos
Ácidos Borônicos/química , Ouro/química , Indóis/química , Nanopartículas Metálicas/química , Polímeros/química , Prata/química , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/isolamento & purificação , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Shigella dysenteriae/imunologia , Shigella dysenteriae/isolamento & purificação , Análise Espectral Raman , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificação
14.
PLoS Pathog ; 16(4): e1008443, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32343740

RESUMO

Staphylococcus aureus (SA) is the causative agent of both skin/soft tissue infections as well as invasive bloodstream infections. Though vaccines have been developed to target both humoral and T cell-mediated immune responses against SA, they have largely failed due to lack of protective efficacy. Group 1 CD1-restricted T cells recognize lipid rather than peptide antigens. Previously found to recognize lipids derived from cell wall of Mycobacterium tuberculosis (Mtb), these cells were associated with protection against Mtb infection in humans. Using a transgenic mouse model expressing human group 1 CD1 molecules (hCD1Tg), we demonstrate that group 1 CD1-restricted T cells can recognize SA-derived lipids in both immunization and infection settings. Systemic infection of hCD1Tg mice showed that SA-specific group 1 CD1-restricted T cell response peaked at 10 days post-infection, and hCD1Tg mice displayed significantly decreased kidney pathology at this time point compared with WT control mice. Immunodominant SA lipid antigens recognized by group 1 CD1-restricted T cells were comprised mainly of cardiolipin and phosphatidyl glycerol, with little contribution from lysyl-phosphatidyl glycerol which is a unique bacterial lipid not present in mammals. Group 1 CD1-restricted T cell lines specific for SA lipids also conferred protection against SA infection in the kidney after adoptive transfer. They were further able to effectively control SA replication in vitro through direct antigen presentation by group 1 CD1-expressing BMDCs. Together, our data demonstrate a previously unknown role for group 1 CD1-restricted SA lipid-specific T cells in the control of systemic MRSA infection.


Assuntos
Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD1/genética , Antígenos CD1/imunologia , Humanos , Imunização , Rim/imunologia , Rim/microbiologia , Lipídeos/imunologia , Camundongos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia
15.
Immunity ; 52(4): 591-605.e6, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294405

RESUMO

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.


Assuntos
Endorribonucleases/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , RNA Bacteriano/metabolismo , RNA de Protozoário/metabolismo , Receptor 8 Toll-Like/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Endorribonucleases/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Escherichia coli/química , Escherichia coli/imunologia , Edição de Genes/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/imunologia , Monócitos/microbiologia , Monócitos/parasitologia , Neutrófilos/microbiologia , Neutrófilos/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Cultura Primária de Células , Estabilidade de RNA , RNA Bacteriano/imunologia , RNA de Protozoário/imunologia , Serratia marcescens/química , Serratia marcescens/imunologia , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Streptococcus/química , Streptococcus/imunologia , Células THP-1 , Receptor 8 Toll-Like/imunologia
16.
Talanta ; 212: 120797, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113559

RESUMO

We report here sensitive photoelectrochemical immunosensing of Staphylococcus aureus (S. aureus) using ZnS-Ag2S/polydopamine (PDA) as a novel photoelectric material and Cu2O as the peroxidase mimic tag. ZnS-Ag2S heterojunctions were prepared on indium tin oxide (ITO) via electrodeposition of ZnS nanoparticles, followed by silver ion exchange. To prepare a PDA/ZnS-Ag2S/ITO, the ZnS-Ag2S/ITO electrode was coated with PDA by self-polymerization of dopamine. The photocurrent of the PDA/ZnS-Ag2S/ITO is 1.55 times that of the ZnS-Ag2S/ITO and 7.87 times that of the ZnS/ITO, indicating a high-performance photoelectric material. A sandwiched-type photoelectrochemical immunosensor was constructed by using PDA/ZnS-Ag2S/ITO as the photoelectrode and Cu2O nanocubes as the labels. Cu2O nanocubes can serve as peroxidase mimic to generate catalytic precipitates on the immunoelectrodes, and both the Cu2O nanocubes and the generated precipitates can decrease the photocurrents of the immunoelectrodes, so a photoelectrochemical immunosensor for detecting S. aureus was constructed, showing a linear range between 10 and 107 CFU mL-1 and a low detection limit of 2 CFU mL-1. Owing to the signal amplification of Cu2O labeling, the sensitivity of the Cu2O-labeled immunosensor is 4 times that of a label-free immunosensor for detecting S. aureus, and the detection limit (2 CFU mL-1) is lower than that of a label-free immunosensor (10 CFU mL-1). This work not only provides a new and efficient photoelectric material but also demonstrated an efficient signal-amplification strategy for photoelectrochemical biosensing.


Assuntos
Cobre/química , Imunoensaio/métodos , Indóis/química , Nanopartículas Metálicas/química , Polímeros/química , Staphylococcus aureus/isolamento & purificação , Anticorpos Imobilizados/imunologia , Técnicas Biossensoriais/métodos , Catálise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Indóis/efeitos da radiação , Luz , Limite de Detecção , Nanopartículas Metálicas/efeitos da radiação , Polímeros/efeitos da radiação , Compostos de Prata/química , Compostos de Prata/efeitos da radiação , Staphylococcus aureus/imunologia , Sulfetos/química , Sulfetos/efeitos da radiação , Compostos de Estanho/química , Compostos de Zinco/química , Compostos de Zinco/efeitos da radiação
17.
J Dairy Sci ; 103(5): 4588-4605, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32113759

RESUMO

Staphylococcus aureus is one of the pathogens most frequently isolated from cases of mastitis worldwide. To decrease the effect of S. aureus mastitis in dairy farming, alternative strategies for controlling mastitis are needed that depend on a better knowledge of cow-to-cow variations in S. aureus antibody production. The present study sought to explore the diversity of S. aureus antibodies produced by dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine. We obtained protein extracts from S. aureus isolates derived from persistent subclinical mastitis. Proteins were fractionated using 2-dimensional gel electrophoresis and Western blotting. Then, Western blotting membranes were exposed to sera from 24 dairy cows that had been divided into the following groups: vaccinated dairy cows that were infected with S. aureus, further subdivided according to whether they (a) remained infected by S. aureus or (b) recovered from the intramammary infection; unvaccinated dairy cows infected with S. aureus; and vaccinated healthy dairy cows with no history of S. aureus mastitis. Proteins found to be reactive by Western blot were identified by mass spectrometry (MALDI/TOF-TOF). Our most important finding was that F0F1 ATP synthase subunit α, succinyl-diaminopimelate desuccinylase, and cysteinyl-tRNA synthetase were potential candidate proteins for the prevention of S. aureus mastitis. This study strengthens the notion that variations among animals should not be ignored and shows that the heterogeneity of antibody production against anti-staphylococcal antigens in animals may enable the identification of new immunotherapy targets.


Assuntos
Anticorpos Antibacterianos/sangue , Mastite Bovina/imunologia , Infecções Estafilocócicas/veterinária , Vacinas Antiestafilocócicas/administração & dosagem , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Bovinos , Feminino , Humanos , Mastite Bovina/microbiologia , Mastite Bovina/prevenção & controle , Leite , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/imunologia
18.
PLoS One ; 15(3): e0229908, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155184

RESUMO

We have previously shown that changes in the microbiome influence how the healing tendon responds to different treatments. The aim of this study was to investigate if changes in the microbiome influence the response to mechanical loading during tendon healing. 90 Sprague-Dawley rats were used. Specific Opportunist and Pathogen Free (SOPF) rats were co-housed with Specific Pathogen Free (SPF) rats, carrying Staphylococcus aureus and other opportunistic microbes. After 6 weeks of co-housing, the SOPF rats were contaminated which was confirmed by Staphylococcus aureus growth. Clean SOPF rats were used as controls. The rats were randomized to full loading or partial unloading by Botox injections in their calf muscles followed by complete Achilles tendon transection. Eight days later, the healing tendons were tested mechanically. The results were analysed by a 2-way ANOVA with interaction between loading and contamination on peak force as the primary outcome and there was an interaction for both peak force (p = 0.049) and stiffness (p = 0.033). Furthermore, partial unloading had a profound effect on most outcome variables. In conclusion, the response to mechanical loading during tendon healing is influenced by changes in the microbiome. Studies aiming for clinical relevance should therefore consider the microbiome of laboratory animals.


Assuntos
Tendão do Calcâneo/lesões , Fenômenos Biomecânicos/imunologia , Modelos Animais de Doenças , Microbiota/imunologia , Cicatrização/imunologia , Animais , Feminino , Humanos , Infecções Oportunistas/imunologia , Infecções Oportunistas/microbiologia , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Estresse Mecânico
19.
Sci Adv ; 6(4): eaax8820, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32010784

RESUMO

Extreme pathophysiological stressors induce expansion of otherwise infrequent leukocyte populations. Here, we found a previously unidentified CD11b+Gr-1+ myeloid cell population that expresses stem cell antigen-1 (Sca-1) induced upon experimental infection with Staphylococcus aureus. Although CD11b+Gr-1+Sca-1+ cells have impaired migratory capacity and superoxide anion-producing activity, they secrete increased levels of several cytokines and chemokines compared to Sca-1- counterparts. The generation of CD11b+Gr-1+Sca-1+ cells is dependent on IFN-γ in vivo, and in vitro stimulation of bone marrow cells or granulocyte-macrophage progenitors with IFN-γ generated CD11b+Gr-1+Sca-1+ cells. Depletion of CD11b+Gr-1+Sca-1+ cells by administrating anti-Sca-1 antibody strongly increased survival rates in an S. aureus infection model by reducing organ damage and inflammatory cytokines. However, adoptive transfer of CD11b+Gr-1+Sca-1+ cells decreased survival rates by worsening the pathogenesis of S. aureus infection. Together, we found a previously unidentified pathogenic CD11b+Gr-1+Sca-1+ population that plays an essential role in mortality during bacterial infection.


Assuntos
Infecções Bacterianas/etiologia , Infecções Bacterianas/mortalidade , Biomarcadores , Células Mieloides/imunologia , Transferência Adotiva , Animais , Antígenos Ly/metabolismo , Infecções Bacterianas/metabolismo , Antígeno CD11b/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Células Mieloides/metabolismo , Fenótipo , Prognóstico , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/mortalidade , Staphylococcus aureus/imunologia , Superóxidos/metabolismo
20.
PLoS One ; 15(2): e0228751, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049976

RESUMO

BACKGROUND: Primary cutaneous CD30+ lymphoproliferative disorders (CD30CLPD) are the second most common type of cutaneous T cell lymphoma (CTCL) and include lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large cell lymphoma (pcALCL). Case reports and small patient series suggest an association of CD30CLPD with atopic disorders. However, the prevalence of atopy in patients with CD30CLPD in retrospective studies depends on patients' recall which is not always reliable. More objective criteria of atopy include evidence of skin reactivity to allergens (positive prick test) and evidence of allergen-specific IgE in serum. This study was undertaken to test the hypothesis that atopy is prevalent in patients with CD30CLPD using serologic criteria of allergen-specific IgE antibodies to aeroallergens and Staphylococcal aureus enterotoxin superantigens (SSAgs). METHODS: We tested serum samples of CD30CLPD for common IgE-specific airborne allergens with the Phadiatop test, which if positive, is regarded as serologic evidence of atopy in adults. Sera were also tested for IgE antibodies reactive to three Staphylococcal enterotoxins with superantigenic properties (SSAg-IgE). Control sera were obtained from adult subjects evaluated for rhino-sinusitis and a negative Phadiatop test. Patients' history of an atopic disorder was obtained by retrospective chart review. FINDINGS: Nearly 50% of patients with the most common LyP types (A and C) had a positive Phadiatop test for allergic sensitization to common airborne allergens, and total serum IgE (IgE-t) was increased compared to non-atopic controls. At the IgE antibody concentration generally used to define serologic atopy (≥ 0.35 kUA/L), 8/31 (26%) samples of CD30CLPD and 7/28 (25%) samples of LyP were reactive to at least one SSAg-IgE compared to 3/52 (6%) control specimens (P = 0.016 and P = 0.028, respectively). TSST1-IgE was detected in 7 (23%) specimens of CD30CLPD, often together with SEB-IgE; SEA-IgE ≥ 0.35 kUA/L was not detected. For control specimens, TSST1-IgE exceeded the 0.35 kUA/L threshold in 3 (6%) specimens. CONCLUSIONS: Patients with LyP types A and C have serologic evidence of atopy against common airborne antigens and SSAgs when compared to control adult subjects who had rhino-sinusitis and a negative Phadiatop test for aero-IgEs. Serologic evidence of atopy exceeded that determined by LyP patients' personal history. The findings support our hypothesis that an atopic diathesis may contribute to the pathogenesis of the most common types of LyP (A and C).


Assuntos
Antígenos de Bactérias/imunologia , Papulose Linfomatoide/imunologia , Neoplasias Cutâneas/imunologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Adolescente , Corticosteroides/farmacologia , Adulto , Idoso , Feminino , Humanos , Imunoglobulina E/imunologia , Papulose Linfomatoide/sangue , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Neoplasias Cutâneas/sangue , Fumar , Adulto Jovem
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