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Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.
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Virus del Dengue , Dengue , Proteínas no Estructurales Virales , Humanos , Citocinas , FN-kappa B/metabolismo , Serogrupo , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND: Monocyte activation is a driver of inflammation in the course of chronic HIV infection. Prostaglandin E2 (PGE2) is known to mediate anti-inflammatory effects, notably the inhibition of tumor necrosis factor- (TNF-) production by monocytes. We aim to investigate the effects of PGE2 on activation of monocytes in chronic HIV infection and the mechanisms through which PGE2 modulates their inflammatory signature. METHODS: We recruited a group of people with HIV (PWH) and matched healthy uninfected persons. We compared plasma levels of PGE2, monocyte activation, and sensitivity of monocytes to the inhibitory actions mediated by PGE2. RESULTS: We found increased plasma levels of PGE2 in PWH, and an activated phenotype in circulating monocytes, compared with uninfected individuals. Monocytes from PWH showed a significant resistance to the inhibitory actions mediated by PGE2; the concentration of PGE2 able to inhibit 50 of the production of TNF- by lipopolysaccharide-stimulated monocytes was 10 times higher in PWH compared with uninfected controls. Furthermore, the expression of phosphodiesterase 4B, a negative regulator of PGE2 activity, was significantly increased in monocytes from PWH. CONCLUSIONS: Resistance to the inhibitory actions mediated by PGE2 could account, at least in part, for the inflammatory profile of circulating monocytes in PWH.
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Dinoprostona , Infecciones por VIH , Humanos , Dinoprostona/metabolismo , Monocitos/metabolismo , Infecciones por VIH/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Expresión Génica , LipopolisacáridosRESUMEN
Severe COVID-19 is associated with a systemic inflammatory response and progressive CD4+ T-cell lymphopenia and dysfunction. We evaluated whether platelets might contribute to CD4+ T-cell dysfunction in COVID-19. We observed a high frequency of CD4+ T cell-platelet aggregates in COVID-19 inpatients that inversely correlated with lymphocyte counts. Platelets from COVID-19 inpatients but not from healthy donors (HD) inhibited the upregulation of CD25 expression and tumour necrosis factor (TNF)-α production by CD4+ T cells. In addition, interferon (IFN)-γ production was increased by platelets from HD but not from COVID-19 inpatients. A high expression of PD-L1 was found in platelets from COVID-19 patients to be inversely correlated with IFN-γ production by activated CD4+ T cells cocultured with platelets. We also found that a PD-L1-blocking antibody significantly restored platelets' ability to stimulate IFN-γ production by CD4+ T cells. Our study suggests that platelets might contribute to disease progression in COVID-19 not only by promoting thrombotic and inflammatory events, but also by affecting CD4+ T cells functionality.
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Antígeno B7-H1 , COVID-19 , Antígeno B7-H1/metabolismo , Plaquetas/metabolismo , Linfocitos T CD4-Positivos , Humanos , Interferón gammaRESUMEN
Severe coronavirus disease 2019 (COVID-19) is associated with an overactive inflammatory response mediated by macrophages. Here, we analyzed the phenotype and function of neutrophils in patients with COVID-19. We found that neutrophils from patients with severe COVID-19 express high levels of CD11b and CD66b, spontaneously produce CXCL8 and CCL2, and show a strong association with platelets. Production of CXCL8 correlated with plasma concentrations of lactate dehydrogenase and D-dimer. Whole blood assays revealed that neutrophils from patients with severe COVID-19 show a clear association with immunoglobulin G (IgG) immune complexes. Moreover, we found that sera from patients with severe disease contain high levels of immune complexes and activate neutrophils through a mechanism partially dependent on FcγRII (CD32). Interestingly, when integrated in immune complexes, anti-severe acute respiratory syndrome coronavirus 2 IgG antibodies from patients with severe COVID-19 displayed a higher proinflammatory profile compared with antibodies from patients with mild disease. Our study suggests that IgG immune complexes might promote the acquisition of an inflammatory signature by neutrophils, worsening the course of COVID-19.
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Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , COVID-19/inmunología , Inmunoglobulina G/inmunología , Activación Neutrófila/inmunología , Adulto , Anciano , Anticuerpos Antivirales/sangre , Complejo Antígeno-Anticuerpo/sangre , Antígenos CD/inmunología , Antígeno CD11b/inmunología , Moléculas de Adhesión Celular/inmunología , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunoglobulina G/sangre , Interleucina-8/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Receptores de IgG/inmunología , SARS-CoV-2/inmunología , Adulto JovenRESUMEN
The main Enterobacteriaceae habitat is the mammal gastrointestinal tract. In most cases, this group of species displays a symbiotic relationship with its hosts. However, some groups may be pathogenic to humans, such as Shiga toxin-producing Escherichia coli and enteroaggregative Escherichia coli. The presence of these groups represents a direct risk to consumers, and recent serotypes displaying the presence of pathogenic genes in both groups are a novel challenge for food production. Thus, microbiological control strategies presenting accurate detection methodologies are required. However, with the appearance of mutations among different species, knowledge, genetic monitoring, and bioinformatics techniques must be expanded. In addition, as a strategy to ensure safe products on an industrial scale, the monitoring by different techniques and fundamentals should be applied throughout the entire processing chain. Therefore, the aim of this review is to describe the pathogenesis mechanisms of different groups, mutant strain dispersion, and current and alternative epidemiological investigation methods.
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Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/epidemiología , Escherichia coli/patogenicidad , Enfermedades Transmitidas por los Alimentos/microbiología , Escherichia coli Shiga-Toxigénica/patogenicidad , Animales , Brotes de Enfermedades/prevención & control , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Inocuidad de los Alimentos/métodos , Enfermedades Transmitidas por los Alimentos/epidemiología , Humanos , Mutación , Filogenia , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , VirulenciaRESUMEN
Staphylococcus pseudintermedius, which is part of the skin microbiome of dogs, causes a variety of opportunistic infections. These infections may become more difficult to treat due to the formation of biofilm. The capacity of S. pseudintermedius to form biofilm, as well as the associated genes, has not been elucidated. This study evaluated the production and composition of S. pseudintermedius biofilm. Samples were collected from both infected dogs and asymptomatic dogs. Isolates were identified using mass spectrometry and Multiplex-PCR. Biofilm production and composition were assessed using a quantitative microtiter plate assay. The presence of ica operon genes and sps genes was investigated using conventional PCR. The investigation of Agr type and virulence genes was conducted in silico on 24 sequenced samples. All strains could produce strong biofilms, with most of the isolates presenting a polysaccharide biofilm. 63.6% of the isolates carried the complete ica operon (ADBC). All samples showed the presence of the genes spsK, spsA, and spsL, while the distribution of other genes varied. Agr type III was the most prevalent (52.2%). All sequenced samples carried the cytotoxins hlb, luk-S, luk-F, as well as the exfoliative toxins siet and se_int. No isolate displayed other exfoliative toxins. Only LB1733 presented a set of different enterotoxins (sea, seb, sec_canine, seh, sek, sel, and seq). Our findings suggest that S. pseudintermedius is a strong producer of biofilm and carries virulence genes.
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Staphylococcus pseudintermedius is an opportunistic pathogen causing a variety of infections that are difficult to treat, especially because of the development of antimicrobial resistance. It has a clonal distribution around the world. To have a better understanding of the MRSP population, we search the presence of MRSP in colonized or infected dogs. Samples from 99 dogs with infections and 35 from asymptomatic dogs were collected. Isolates were identified by mass spectrometry and Multiplex-PCR. The mecA gene was confirmed by conventional PCR. MRSP strains were analyzed by whole-genome sequencing. 75 S. pseudintermedius were identified, most from infection cases. The species were isolated from 70 out of the 135 dogs. Penicillin and Trimethoprim/Sulfamethoxazole presented higher resistance rates. Forty-seven strains were classified as multi-drug resistant (MDR), and were more isolated from dogs with infection (P < 0.05). Eighteen samples were classified as MRSP, representing 24.0% of the population. Six of 16 MRSP sequenced samples belonged to the world spread clone ST71; others belonged to unknown clones. Most samples carried the SCCmec type IIIA. Twenty-one different genetic resistance determinants were found among MRPS strains. MRSP is circulating among infected and colonized dogs in Rio de Janeiro, Brazil.
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Enfermedades de los Perros , Infecciones Estafilocócicas , Perros , Animales , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/epidemiología , Antibacterianos/farmacología , Resistencia a la Meticilina , Brasil , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/epidemiología , Reacción en Cadena de la Polimerasa Multiplex , Variación Genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Coronavirus infection in humans is usually associated to respiratory tract illnesses, ranging in severity from mild to life-threatening respiratory failure. The aryl hydrocarbon receptor (AHR) was recently identified as a host factor for Zika and dengue viruses; AHR antagonists boost antiviral immunity, decrease viral titers and ameliorate Zika-induced pathology in vivo. Here we report that AHR is activated by infection with different coronaviruses, potentially impacting antiviral immunity and lung epithelial cells. Indeed, the analysis of single-cell RNA-seq from lung tissue detected increased expression of AHR and AHR transcriptional targets, suggesting AHR signaling activation in SARS-CoV-2-infected epithelial cells from COVID-19 patients. Moreover, we detected an association between AHR expression and viral load in SARS-CoV-2 infected patients. Finally, we found that the pharmacological inhibition of AHR suppressed the replication in vitro of one of the causative agents of the common cold, HCoV-229E, and the causative agent of the COVID-19 pandemic, SARS-CoV-2. Taken together, these findings suggest that AHR activation is a common strategy used by coronaviruses to evade antiviral immunity and promote viral replication, which may also contribute to lung pathology. Future studies should further evaluate the potential of AHR as a target for host-directed antiviral therapy.
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Infecciones por Coronavirus/metabolismo , Coronavirus/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Femenino , Humanos , Masculino , Receptores de Hidrocarburo de Aril/genética , SARS-CoV-2/fisiologíaRESUMEN
S. schleiferi is one of the main species isolated from canine otitis externa, pyoderma and from apparently healthy dogs. The species is divided into two subspecies, S. schleiferi schleiferi and S. schleiferi coagulans. MALDI-TOF MS does not distinguish correctly these two subspecies. This study aimed to identify biomarkers that could possibly discriminate Staphylococcus schleiferi subspecies by MALDI-TOF MS. Twelve strains (eight S. schleiferi schleiferi and four S. schleiferi coagulans) were firstly identified. Each isolate was submitted to a protein extraction protocol and subjected to spectrometry on Bruker Microflex LT mass spectrometer. Spectra were analyzed with the BioNumerics software v7.6. Our results showed that spectra clustered according to subspecies, and a set of five MALDI-TOF MS biomarkers were selected to enable the discrimination of S. schleiferi subspecies. In addition, these biomarkers were predicted to represent highly conserved proteins, which could contribute to the identification of subspecies-specific proteins that could be used for improved subspecies identification in clinical samples.
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Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus/clasificación , Animales , Biomarcadores/análisis , Perros , Estudios Prospectivos , Reproducibilidad de los Resultados , Staphylococcus/aislamiento & purificaciónRESUMEN
Inflammatory dendritic cells (DCs) are a distinct subset of DCs that derive from circulating monocytes infiltrating injured tissues. Monocytes can differentiate into DCs with different functional signatures, depending on the presence of environment stimuli. Among these stimuli, transforming growth factor-beta (TGF-ß) and prostaglandin E2 (PGE2) have been shown to modulate the differentiation of monocytes into DCs with different phenotypes and functional profiles. In fact, both mediators lead to contrasting outcomes regarding the production of inflammatory and anti-inflammatory cytokines. Previously, we have shown that human semen, which contains high concentrations of PGE2, promoted the differentiation of DCs into a tolerogenic profile through a mechanism dependent on signaling by E-prostanoid receptors 2 and 4. Notably, this effect was induced despite the huge concentration of TGF-ß present in semen, suggesting that PGE2 overrides the influence exerted by TGF-ß. No previous studies have analyzed the joint actions induced by PGE2 and TGF-ß on the function of monocytes or DCs. Here, we analyzed the phenotype and functional profile of monocyte-derived DCs differentiated in the presence of TGF-ß and PGE2. DC differentiation guided by TGF-ß alone enhanced the expression of CD1a and abrogated LPS-induced expression of IL-10, while differentiation in the presence of PGE2 impaired CD1a expression, preserved CD14 expression, abrogated IL-12 and IL-23 production, stimulated IL-10 production, and promoted the expansion of FoxP3+ regulatory T cells in a mixed lymphocyte reaction. Interestingly, DCs differentiated in the presence of TGF-ß and PGE2 showed a phenotype and functional profile closely resembling those induced by PGE2 alone. Finally, we found that PGE2 inhibited TGF-ß signaling through an action exerted by EP2 and EP4 receptors coupled to cyclic AMP increase and protein kinase A activity. These results indicate that PGE2 suppresses the influence exerted by TGF-ß during DC differentiation, imprinting a tolerogenic signature. High concentrations of TGF-ß and PGE2 are usually found in infectious, autoimmune, and neoplastic diseases. Our observations suggest that in these scenarios PGE2 might play a mandatory role in the acquisition of a regulatory profile by DCs.