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Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID-19). While COVID-19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID-19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID-19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID-19 patients requiring mechanical ventilation. BALs from severe COVID-19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4 , LTE4 , and eoxin E4 . Monohydroxylated 15-lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID-19 involves pro- and anti-inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipid mediators.
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COVID-19 , Leucotrieno B4/metabolismo , Leucotrieno E4/análogos & derivados , Leucotrieno E4/metabolismo , Lipoxinas/metabolismo , Pulmón , SARS-CoV-2/metabolismo , Adulto , COVID-19/metabolismo , COVID-19/patología , COVID-19/terapia , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana EdadRESUMEN
A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.
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COVID-19 , Nanopartículas , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Anticuerpos ampliamente neutralizantes , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Ratones , Ratones Endogámicos BALB C , Potexvirus , SARS-CoV-2RESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to a variety of clinical outcomes, ranging from the absence of symptoms to severe acute respiratory disease and ultimately death. A feature of patients with severe coronavirus disease 2019 (COVID-19) is the abundance of inflammatory cytokines in the blood. Elevated levels of cytokines are predictive of infection severity and clinical outcome. In contrast, studies aimed at defining the driving forces behind the inflammation in lungs of subjects with severe COVID-19 remain scarce. OBJECTIVE: Our aim was to analyze and compare the plasma and bronchoalveolar lavage (BAL) fluids of patients with severe COVID-19 (n = 45) for the presence of cytokines and lipid mediators of inflammation (LMIs). METHODS: Cytokines were measured by using Luminex multiplex assay, and LMIs were measured by using liquid chromatography-tandem mass spectrometry. RESULTS: We revealed high concentrations of numerous cytokines, chemokines, and LMIs in the BAL fluid of patients with severe COVID-19. Of the 13 most abundant mediators in BAL fluid, 11 were chemokines, with CXCL1 and CXCL8 being 200 times more abundant than IL-6 and TNF-α. Eicosanoid levels were also elevated in the lungs of subjects with severe COVID-19. Consistent with the presence chemotactic molecules, BAL fluid samples were enriched for neutrophils, lymphocytes, and eosinophils. Inflammatory cytokines and LMIs in plasma showed limited correlations with those present in BAL fluid, arguing that circulating inflammatory molecules may not be a reliable proxy of the inflammation occurring in the lungs of patients with severe COVID-19. CONCLUSIONS: Our findings indicate that hyperinflammation of the lungs of patients with severe COVID-19 is fueled by excessive production of chemokines and eicosanoids. Therapeutic strategies to dampen inflammation in patients with COVID-19 should be tailored accordingly.
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COVID-19/inmunología , Citocinas/inmunología , Eicosanoides/inmunología , Inflamación/inmunología , Pulmón/inmunología , SARS-CoV-2 , Adulto , Anciano , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , COVID-19/sangre , Citocinas/sangre , Femenino , Humanos , Inflamación/sangre , Pulmón/citología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Índice de Severidad de la EnfermedadRESUMEN
Approximately 1% of people worldwide carry a copy of the human herpesvirus 6A or 6B (HHV-6A/B) in every cell of their body. This condition is referred to as inherited chromosomally integrated HHV-6A/B (iciHHV-6A/B). The mechanisms leading to iciHHV-6A/B chromosomal integration are yet to be identified. A recent report suggested that the rs73185306 C/T single-nucleotide polymorphism (SNP) represents a favorable predisposing factor leading to HHV-6A/B integration. After genotype analysis of an independent cohort (N = 11 967), we report no association between the rs73185306 C/T SNP and HHV-6A/B chromosomal integration (odds ratio, 0.90 [95% confidence interval, .54-1.51]; P = .69).
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Predisposición Genética a la Enfermedad , Herpesvirus Humano 6/genética , Mutación Puntual , Estudios de Casos y Controles , Estudios de Cohortes , ADN Viral/genética , Humanos , Reproducibilidad de los Resultados , Factores de Riesgo , Integración ViralRESUMEN
Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carries one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response are still unclear. In this study, we screened DNA sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A- and 4 iciHHV-6B-positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A- and iciHHV-6B-positive individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age- and sex-matched controls were analyzed for antibodies to control antigens (cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza virus [FLU]) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) than do non-iciHHV-6-positive individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57, and U72, were identical between controls and iciHHV-6A/B+ subjects. CMV-seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150 than do CMV-seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B-specific antibody response.IMPORTANCE HHV-6A and -6B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world's population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression data set, we showed that the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate data sets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks, and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.
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Anticuerpos Antivirales/sangre , Cromosomas Humanos/química , Herpesvirus Humano 6/genética , ARN Viral/genética , Infecciones por Roseolovirus/virología , Glándulas Suprarrenales/inmunología , Glándulas Suprarrenales/virología , Anciano , Encéfalo/inmunología , Encéfalo/virología , Estudios de Cohortes , Citomegalovirus/inmunología , Esófago/inmunología , Esófago/virología , Femenino , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 6/clasificación , Herpesvirus Humano 6/inmunología , Humanos , Patrón de Herencia , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Orthomyxoviridae/inmunología , Filogenia , ARN Viral/inmunología , Infecciones por Roseolovirus/genética , Infecciones por Roseolovirus/inmunología , Testículo/inmunología , Testículo/virología , Integración Viral , Secuenciación Completa del GenomaRESUMEN
Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.
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Herpesvirus Humano 6/fisiología , Cultivo de Virus/métodos , Integración Viral , Técnicas de Cultivo de Célula/métodos , Línea Celular , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Inherited chromosomally integrated human herpesvirus-6 (iciHHV-6) results in the germ-line transmission of the HHV-6 genome. Every somatic cell of iciHHV-6+ individuals contains the HHV-6 genome integrated in the telomere of chromosomes. Whether having iciHHV-6 predisposes humans to diseases remains undefined. DNA from 19,597 participants between 40 and 69 years of age were analyzed by quantitative PCR (qPCR) for the presence of iciHHV-6. Telomere lengths were determined by qPCR. Medical records, hematological, biochemical, and anthropometric measurements and telomere lengths were compared between iciHHV-6+ and iciHHV-6- subjects. The prevalence of iciHHV-6 was 0.58%. Two-way ANOVA with a Holm-Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on continuous outcomes. Two-way logistic regression with a Holm-Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on disease prevalence. Of 50 diseases monitored, a single one, angina pectoris, is significantly elevated (3.3×) in iciHHV-6+ individuals relative to iciHHV-6- subjects (P = 0.017; 95% CI, 1.73-6.35). When adjusted for potential confounding factors (age, body mass index, percent body fat, and systolic blood pressure), the prevalence of angina remained three times greater in iciHHV-6+ subjects (P = 0.015; 95%CI, 1.23-7.15). Analyses of telomere lengths between iciHHV-6- without angina, iciHHV-6- with angina, and iciHHV-6+ with angina indicate that iciHHV-6+ with angina have shorter telomeres than age-matched iciHHV-6- subjects (P = 0.006). Our study represents, to our knowledge, the first large-scale analysis of disease association with iciHHV-6. Our results are consistent with iciHHV-6 representing a risk factor for the development of angina.
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Angina de Pecho/virología , Predisposición Genética a la Enfermedad , Herpesvirus Humano 6/genética , Adulto , Anciano , Angina de Pecho/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , TelómeroRESUMEN
Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3' to 5' exonuclease activity on dsDNA with a preference for 3'-recessed ends. Once the DNA strand reaches 8-10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3' end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.
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Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/química , Exodesoxirribonucleasas/metabolismo , Herpesvirus Humano 6/enzimología , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/química , ADN Helicasas/química , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/química , Unión Proteica , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
Platelets play crucial functions in hemostasis and the prevention of bleeding. During H1N1 influenza A virus infection, platelets display activation markers. The platelet activation triggers during H1N1 infection remain elusive. We observed that H1N1 induces surface receptor activation, lipid mediator synthesis, and release of microparticles from platelets. These activation processes require the presence of serum/plasma, pointing to the contribution of soluble factor(s). Considering that immune complexes in the H1N1 pandemic were reported to play a pathogenic role, we assessed their contribution in H1N1-induced platelet activation. In influenza-immunized subjects, we observed that the virus scaffolds with immunoglobulin G (IgG) to form immune complexes that promote platelet activation. Mechanistically, this activation occurs through stimulation of low-affinity type 2 receptor for Fc portion of IgG (FcγRIIA), a receptor for immune complexes, independently of thrombin. Using a combination of in vitro and in vivo approaches, we found that the antibodies from H3N2-immunized mice activate transgenic mouse platelets that express FcγRIIA when put in the presence of H1N1, suggesting that cross-reacting influenza antibodies suffice. Alternatively, H1N1 can activate platelets via thrombin formation, independently of complement and FcγRIIA. These observations identify both the adaptive immune response and the innate response against pathogens as 2 intertwined processes that activate platelets during influenza infections.
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Plaquetas/inmunología , Plaquetas/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Activación Plaquetaria/inmunología , Receptores de IgG/metabolismo , Transducción de Señal , Trombina/metabolismo , Animales , Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Humanos , Inmunidad Innata , Inmunofenotipificación , Ratones , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Fenotipo , Receptores de IgG/genéticaRESUMEN
Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T-cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV-6 infections, in part due to a lack of identified protective CD8(+) T-cell epitopes. Our goal was to identify CD8(+) T-cell viral epitopes derived from the HHV-6B immediate-early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA-A*02, HLA-A*03, and HLA-B*07. These epitopes were functionally tested for their ability to induce CD8(+) T-cell expansion and kill HHV-6-infected autologous cells. Cross-reactivity of specific HHV-6B-expanded T cells against HHV-6A-infected cells was also confirmed for a conserved epitope presented by HLA-A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV-6 reactivation in BMT recipients.
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Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Antígeno HLA-A3/inmunología , Antígeno HLA-B7/inmunología , Herpesvirus Humano 6/inmunología , Proteínas Inmediatas-Precoces/inmunología , Adolescente , Traslado Adoptivo , Adulto , Animales , Linfocitos T CD8-positivos/patología , Proliferación Celular/genética , Epítopos de Linfocito T/genética , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A3/genética , Antígeno HLA-B7/genética , Herpesvirus Humano 6/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Inmunidad Celular/genética , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Infecciones por Roseolovirus/genética , Infecciones por Roseolovirus/inmunología , Infecciones por Roseolovirus/patología , Infecciones por Roseolovirus/terapiaRESUMEN
The scientific and medical community faced an unprecedented global health hazard that led to nearly 7 million deaths attributable to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In spite of the development of efficient vaccines against SARS-CoV-2, many people remain at risk of developing severe symptoms as the virus continues to spread without beneficial patient therapy. The hyper-inflammatory response to SARS-CoV-2 infection progressing to acute respiratory distress syndrome remains an unmet medical need for improving patient care. The viral infection stimulates alveolar macrophages to adopt an inflammatory phenotype regulated, at least in part, by the cluster of differentiation 36 receptor (CD36) to produce unrestrained inflammatory cytokine secretions. We suggest herein that the modulation of the macrophage response using the synthetic CD36 ligand hexarelin offers potential as therapy for halting respiratory failure in SARS-CoV-2-infected patients.
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ABSTRACT: Megakaryocytes (MKs), integral to platelet production, predominantly reside in the bone marrow (BM) and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence MK transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection could affect the transcriptome of BM MKs. Using spatial transcriptomics to discriminate subpopulations of MKs based on proximity to BM sinusoids, we identified â¼19 000 genes in MKs. Machine learning techniques revealed that the transcriptome of healthy murine BM MKs exhibited minimal differences based on proximity to sinusoid vessels. Furthermore, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, MKs were not significantly different from those from healthy mice. Conversely, a significant divergence in the MK transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in the BM, and it was no longer detectable in the lungs. Under these conditions, the MK transcriptional landscape was enriched in pathways associated with histone modifications, MK differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type I interferon signature and calprotectin (S100A8/A9) were not induced in MKs under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the BM, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of BM MKs may emerge through COVID-19-related pathogenesis.
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Médula Ósea , COVID-19 , Megacariocitos , SARS-CoV-2 , Transcriptoma , COVID-19/patología , COVID-19/virología , COVID-19/genética , COVID-19/metabolismo , Megacariocitos/metabolismo , Megacariocitos/virología , Animales , SARS-CoV-2/fisiología , SARS-CoV-2/genética , Ratones , Médula Ósea/metabolismo , Médula Ósea/patología , Calgranulina B/metabolismo , Calgranulina B/genética , Humanos , Calgranulina A/metabolismo , Calgranulina A/genética , Modelos Animales de EnfermedadRESUMEN
The persistence of SARS-CoV-2 despite the development of vaccines and a degree of herd immunity is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of wild-type (WT) SARS-CoV-2 in Balb/c mice yield mouse-adapted strains with greater infectivity and mortality. We investigate if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing the human ACE2 receptor, in a BSL-3 laboratory without selective pressures, drives human health-relevant evolution and if evolution is lineage-dependent. Late-passage virus causes more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurs with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, is co-inherited at times with spike E1182G per Nanopore sequencing, existing in different within-sample viral variants at others. Both S371F and E1182G are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2's tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribution.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/genética , SARS-CoV-2/genética , Ratones Endogámicos BALB C , MamíferosRESUMEN
COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1ß and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNß expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNß promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.
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COVID-19 , FN-kappa B , Animales , Humanos , Ratones , Antivirales , Inflamasomas , Inflamación , SARS-CoV-2RESUMEN
Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Ratones , Activación Plaquetaria , Trombina , Tromboplastina/metabolismoRESUMEN
Coronavirus disease 19 (COVID-19) is the clinical manifestation of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. A hallmark of COVID-19 is a lung inflammation characterized by an abundant leukocyte infiltrate, elevated levels of cytokines/chemokines, lipid mediators of inflammation (LMI) and microthrombotic events. Animal models are useful for understanding the pathophysiological events leading to COVID-19. One such animal model is the K18-ACE2 transgenic mice. Despite their importance in inflammation, the study of LMI in lung of SARS-CoV-2 infected K18-ACE2 mice has yet to be studied to our knowledge. Using tandem mass spectrometry, the lung lipidome at different time points of infection was analyzed. Significantly increased LMI included N-oleoyl-serine, N-linoleoyl-glycine, N-oleoyl-alanine, 1/2-linoleoyl-glycerol, 1/2-docosahexaenoyl-glycerol and 12-hydroxy-eicosapenatenoic acid. The levels of prostaglandin (PG) E1, PGF2α, stearoyl-ethanolamide and linoleoyl-ethanolamide were found to be significantly reduced relative to mock-infected mice. Other LMI were present at similar levels (or undetected) in both uninfected and infected mouse lungs. In parallel to LMI measures, transcriptomic and cytokine/chemokine profiling were performed. Viral replication was robust with maximal lung viral loads detected on days 2-3 post-infection. Lung histology revealed leukocyte infiltration starting on day 3 post-infection, which correlated with the presence of high concentrations of several chemokines/cytokines. At early times post-infection, the plasma of infected mice contained highly elevated concentration of D-dimers suggestive of blood clot formation/dissolution. In support, the presence of blood clots in the lung vasculature was observed during infection. RNA-Seq analysis of lung tissues indicate that SARS-CoV-2 infection results in the progressive modulation of several hundred genes, including several inflammatory mediators and genes related to the interferons. Analysis of the lung lipidome indicated modest, yet significant modulation of a minority of lipids. In summary, our study suggests that SARS-CoV-2 infection in humans and mice share common features, such as elevated levels of chemokines in lungs, leukocyte infiltration and increased levels of circulating D-dimers. However, the K18-ACE2 mouse model highlight major differences in terms of LMI being produced in response to SARS-CoV-2 infection. The potential reasons and impact of these differences on the pathology and therapeutic strategies to be employed to treat severe COVID-19 are discussed.
Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Animales , Quimiocinas , Citocinas , Modelos Animales de Enfermedad , Inflamación/patología , Mediadores de Inflamación , Lípidos , Pulmón/patología , Ratones , Ratones TransgénicosRESUMEN
Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8-13) (NT(8-13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8-13). We first examined the binding affinities of this novel NT(8-13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of ß-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article "Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog" published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.
RESUMEN
Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS1 and NTS2 receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8-13) derivative denoted JMV2009, in which the Pro10 residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS1 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS1 and NTS2, improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptive-related behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerve-injured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.
Asunto(s)
Analgésicos/uso terapéutico , Neurotensina/análogos & derivados , Neurotensina/uso terapéutico , Dolor/tratamiento farmacológico , Receptores de Neurotensina/agonistas , Analgésicos/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Masculino , Neurotensina/farmacología , Dolor/fisiopatología , Ratas Sprague-Dawley , Receptores de Neurotensina/fisiologíaRESUMEN
Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are associated with various diseases including seizures and encephalitis. The HHV-6A/B genomes have been shown to be present in an integrated state in the telomeres of latently infected cells. In addition, integration of HHV-6A/B in germ cells has resulted in individuals harboring this inherited chromosomally integrated HHV-6A/B (iciHHV-6) in every cell of their body. Until now, the viral transcriptome and the epigenetic modifications that contribute to the silencing of the integrated virus genome remain elusive. In the current study, we used a patient-derived iciHHV-6A cell line to assess the global viral gene expression profile by RNA-seq, and the chromatin profiles by MNase-seq and ChIP-seq analyses. In addition, we investigated an in vitro generated cell line (293-HHV-6A) that expresses GFP upon the addition of agents commonly used to induce herpesvirus reactivation such as TPA. No viral gene expression including miRNAs was detected from the HHV-6A genomes, indicating that the integrated virus is transcriptionally silent. Intriguingly, upon stimulation of the 293-HHV-6A cell line with TPA, only foreign promoters in the virus genome were activated, while all HHV-6A promoters remained completely silenced. The transcriptional silencing of latent HHV-6A was further supported by MNase-seq results, which demonstrate that the latent viral genome resides in a highly condensed nucleosome-associated state. We further explored the enrichment profiles of histone modifications via ChIP-seq analysis. Our results indicated that the HHV-6 genome is modestly enriched with the repressive histone marks H3K9me3/H3K27me3 and does not possess the active histone modifications H3K27ac/H3K4me3. Overall, these results indicate that HHV-6 genomes reside in a condensed chromatin state, providing insight into the epigenetic mechanisms associated with the silencing of the integrated HHV-6A genome.
RESUMEN
The central administration of neurotensin (NT) or of its C-terminal hexapeptide fragment NT(8-13), produces strong analgesic effects in tests evaluating acute pain. The use of NT-derived peptides as pharmaceutical agents to relief severe pain in patients could be of great interest. Unfortunately, peptides do not readily penetrate the blood-brain barrier. We have observed that the cyclic NT(8-13) analogue, c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012, compound 1), when peripherally administered to mice produced analgesic and hypothermic effects, suggesting the peptide penetrates the blood-brain barrier and functions effectively like a drug. Moreover, dimeric compounds show increased potency compared to their corresponding monomer. We present the synthesis of the cyclic dimer compound 1 (JMV2012). In mice, compound 1 induced a profound hypothermia and a potent analgesia, even when peripherally administered. Compound 1 appears to be an ideal lead compound for the development of bioactive NT analogues as novel analgesics drugs.