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BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.
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Microbioma Gastrointestinal , Doença Granulomatosa Crônica , Doenças Inflamatórias Intestinais , Humanos , Doença Granulomatosa Crônica/genética , NADPH Oxidases , Estudos TransversaisRESUMO
BACKGROUND: Laboratory screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key mitigation measure to avoid the spread of infection among recruits starting basic combat training in a congregate setting. Because viral nucleic acid can be detected persistently after recovery, we evaluated other laboratory markers to distinguish recruits who could proceed with training from those who were infected. METHODS: Recruits isolated for coronavirus disease 2019 (COVID-19) were serially tested for SARS-CoV-2 subgenomic ribonucleic acid (sgRNA), and viral load (VL) by reverse-transcriptase polymerase chain reaction (RT-PCR), and for anti- SARS-CoV-2. Cluster and quadratic discriminant analyses of results were performed. RESULTS: Among 229 recruits isolated for COVID-19, those with a RT-PCR cycle threshold >30.49 (sensitivity 95%, specificity 96%) or having sgRNA log10 RNA copies/mL <3.09 (sensitivity and specificity 96%) at entry into isolation were likely SARS-CoV-2 uninfected. Viral load >4.58 log10 RNA copies/mL or anti-SARS-CoV-2 signal-to-cutoff ratio <1.38 (VL: sensitivity and specificity 93%; anti-SARS-CoV-2: sensitivity 83%, specificity 79%) had comparatively lower sensitivity and specificity when used alone for discrimination of infected from uninfected. CONCLUSIONS: Orthogonal laboratory assays used in combination with RT-PCR may have utility in determining SARS-CoV-2 infection status for decisions regarding isolation.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Teste para COVID-19 , Sensibilidade e Especificidade , RNA , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.
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Complexo Antígeno-Anticorpo/metabolismo , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Macrófagos/imunologia , Biópsia , Diferenciação Celular/imunologia , Linhagem Celular , Progressão da Doença , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Receptores Toll-Like/metabolismo , Adulto JovemRESUMO
Diversity and plasticity are the hallmarks of macrophages. The two most well-defined macrophage subsets are the classically activated macrophages (CAMÏs) and the IL-4-derived alternatively activated macrophages (AAMÏs). Through a series of studies, we previously identified and characterized a distinct population of macrophages with immunoregulatory functions, collectively termed regulatory macrophages (RMÏs). Although considerable advances have been made in understanding these various macrophage subsets, it is not known whether macrophages of one activation state can influence the other. In this study, we examined whether RMÏs capable of inhibiting inflammatory responses of CAMÏs could also inhibit AAMÏs and their profibrotic responses. Our results demonstrated that RMÏs significantly dampened the alternate activation phenotype of AAMÏs generated in vitro and intrinsically occurring AAMÏs from TACI-/- macrophages. Further, RMÏs inhibited AAMÏ-promoted arginase activity and fibroblast proliferation in vitro. This inhibition occurred regardless of the strength, duration, and mode of alternative activation and was only partially dependent on IL-10. In the chlorhexidine gluconate-induced peritoneal fibrosis model, AAMÏs worsened the fibrosis, but RMÏs rescued mice from AAMÏ-mediated pathological conditions. Taken together, our study demonstrates that RMÏs are a specialized subset of macrophages with a nonredundant role in limiting overt proregenerative functions of AAMÏs, a role distinct from their well-defined role of suppression of inflammatory responses by CAMÏs.
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Fibrose/patologia , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Animais , Feminino , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteína Transmembrana Ativadora e Interagente do CAML/deficiênciaRESUMO
In recent years, researchers have devoted much attention to the diverse roles of macrophages and their contributions to tissue development, wound healing, and angiogenesis. What should not be lost in the discussions regarding the diverse biology of these cells is that when perturbed, macrophages are the primary contributors to potentially pathological inflammatory processes. Macrophages stand poised to rapidly produce large amounts of inflammatory cytokines in response to danger signals. The production of these cytokines can initiate a cascade of inflammatory mediator release that can lead to wholesale tissue destruction. The destructive inflammatory capability of macrophages is amplified by exposure to exogenous interferon-γ, which prolongs and heightens inflammatory responses. In simple terms, macrophages can thus be viewed as incendiary devices with hair triggers waiting to detonate. We have begun to ask questions about how these cells can be regulated to mitigate the collateral destruction associated with macrophage activation.
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Inflamação/fisiopatologia , Macrófagos/fisiologia , Animais , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismoRESUMO
Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1ß and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1ß and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (TH17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity.
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Efeito Espectador/imunologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Células Th17/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Efeito Espectador/genética , Proteínas Adaptadoras de Sinalização CARD , Complemento C3a/genética , Complemento C5a/genética , Complexo de Ataque à Membrana do Sistema Complemento/genética , Células HEK293 , Humanos , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fagocitose/genéticaRESUMO
BACKGROUND: Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE: We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS: We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS: We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS: Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.
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Fatores de Transcrição Forkhead/genética , Genes Dominantes , Doenças Genéticas Ligadas ao Cromossomo X/genética , Enteropatias/genética , Mutação , Poliendocrinopatias Autoimunes/genética , Fator de Transcrição STAT1/genética , Adolescente , Autoanticorpos/imunologia , Linhagem Celular Transformada , Criança , Pré-Escolar , DNA/metabolismo , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Imunofenotipagem , Interferon-alfa/imunologia , Interferon gama/farmacologia , Interleucina-17/imunologia , Interleucinas/imunologia , Enteropatias/diagnóstico , Enteropatias/imunologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Fenótipo , Fosforilação/efeitos dos fármacos , Poliendocrinopatias Autoimunes/diagnóstico , Poliendocrinopatias Autoimunes/imunologia , Fator de Transcrição STAT1/metabolismo , Síndrome , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Ativação Transcricional , Interleucina 22RESUMO
BACKGROUND: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.
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Coccidioidomicose/genética , Histoplasmose/genética , Mutação , Fator de Transcrição STAT1/genética , Adolescente , Adulto , Linhagem Celular Transformada , Criança , Coccidioidomicose/diagnóstico , Coccidioidomicose/imunologia , Citocinas/biossíntese , Feminino , Regulação da Expressão Gênica , Histoplasmose/diagnóstico , Histoplasmose/imunologia , Humanos , Masculino , Fosforilação , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator de Transcrição STAT1/metabolismo , Células Th17/imunologia , Ativação Transcricional , Adulto JovemRESUMO
The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.
Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais , Ubiquitina/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Quimiocinas/metabolismo , Quimiotaxia , Dimerização , Endossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Sistema Imunitário/metabolismo , Células Jurkat , Lentivirus/genética , Linfócitos/citologia , Lisossomos/metabolismo , Microscopia Confocal/métodos , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Dietary interventions with bioactive compounds have been found to suppress the accumulation of senescent cells and senescence-associated secretory phenotypes (SASPs). One such compound, curcumin (CUR), has beneficial health and biological effects, including antioxidant and anti-inflammatory properties, but its ability to prevent hepatic cellular senescence is unclear. The objective of this study was to investigate the effects of dietary CUR as an antioxidant on hepatic cellular senescence and determine its benefits on aged mice. We screened the hepatic transcriptome and found that CUR supplementation led to the downregulation of senescence-associated hepatic gene expressions in both usually fed and nutritionally challenged aged mice. Our results showed that CUR supplementation enhanced antioxidant properties and suppressed mitogen-activated protein kinase (MAPK) signaling cascades in the liver, particularly c-Jun N-terminal kinase (JNK) in aged mice and p38 in diet-induced obese aged mice. Furthermore, dietary CUR decreased the phosphorylation of nuclear factor-κB (NF-κB), a downstream transcription factor of JNK and p38, and inhibited the mRNA expression of proinflammatory cytokines and SASPs. The potency of CUR administration was demonstrated in aged mice via enhanced insulin homeostasis along with declined body weight. Taken together, these results suggest that CUR supplementation may be a nutritional strategy to prevent hepatic cellular senescence.
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Introduction: Transcranial Magnetic Stimulation (TMS) is a noninvasive technique that uses pulsed magnetic fields to affect the physiology of the brain and central nervous system. Repetitive TMS (rTMS) has been used to study and treat several neurological conditions, but its complex molecular basis is largely unexplored. Methods: Utilizing three experimental rat models (in vitro, ex vivo, and in vivo) and employing genome-wide microarray analysis, our study reveals the extensive impact of rTMS treatment on gene expression patterns. Results: These effects are observed across various stimulation protocols, in diverse tissues, and are influenced by time and age. Notably, rTMS-induced alterations in gene expression span a wide range of biological pathways, such as glutamatergic, GABAergic, and anti-inflammatory pathways, ion channels, myelination, mitochondrial energetics, multiple neuron-and synapse-specific genes. Discussion: This comprehensive transcriptional analysis induced by rTMS stimulation serves as a foundational characterization for subsequent experimental investigations and the exploration of potential clinical applications.
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BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.
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Fibrose Cística , Mycobacterium abscessus , Mycobacterium , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fibrose Cística/microbiologia , Genômica , Glucose , Pulmão , Mutação , Mycobacterium/genética , Mycobacterium abscessus/genética , Fator de Necrose Tumoral alfa/genética , Porinas/genética , Porinas/metabolismoRESUMO
BACKGROUND: Vaccination protects against severe COVID-19 manifestations. For those with post-COVID-19 conditions (PCC) or long COVID, the impact of COVID-19 vaccination on the evolution of symptoms, immune responses, and viral persistence is unclear. METHODS: In this prospective observational cohort study, we evaluated the number of PCC symptoms, affected organ systems, and psychological well-being scores before and after patients with PCC received COVID-19 vaccination. We simultaneously evaluated biomarkers of systemic inflammation and levels of plasma cytokines/chemokines. We measured plasma and intracellular levels of SARS-CoV-2 antigens, and immunoreactivity to SARS-CoV-2 antigens in blood. RESULTS: COVID-19 vaccination was associated with decreases in number of PCC symptoms (pre-vaccination: 6.56 ± 3.1 vs post-vaccination: 3.92 ± 4.02; P <0.001) and affected organ systems (pre-vaccination: 3.19 ± 1.04 vs post-vaccination: 1.89 ± 1.12; P <0.001), and increases in World Health Organization (WHO)-5 Well-Being Index Scores (pre-vaccination: 42.67 ± 22.76 vs post-vaccination: 56.15 ± 22.83; P <0.001). Patients with PCC also had significantly decreased levels of several pro-inflammatory plasma cytokines/chemokines after COVID-19 vaccination including sCD40L, GRO-âº, macrophage inflammatory protein (MIP)-1âº, interleukin (IL)-12p40, G-colony stimulating factor (CSF), M-CSF, IL-1ß, and stem cell factor (SCF). PCC participants presented a certain level of immunoreactivity toward SARS-CoV-2, that was boosted with vaccination. SARS-CoV-2 S1 antigen persisted in the blood of PCC participants, mostly in non-classical monocytes, regardless of participants receiving vaccination. CONCLUSIONS: Our study shows higher pro-inflammatory responses associated with PCC symptoms and brings forward a possible role for vaccination in mitigating PCC symptoms by decreasing systemic inflammation. We also observed persistence of viral products independent of vaccination that could be involved in perpetuating inflammation through non-classical monocytes.
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COVID-19 , Síndrome de COVID-19 Pós-Aguda , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Estudos Prospectivos , SARS-CoV-2 , Vacinação , Citocinas , Inflamação , Imunidade , QuimiocinasRESUMO
STAT1 is a key component of Interferon (IFN)-γ and IFN-α signaling and mediates protection against mycobacteria, fungal, viral infections, and cancer. Dominant negative inhibitory as well as gain of function heterozygous STAT1 mutations demonstrate that IFN-γ driven cellular responses need to be tightly regulated to control infections. We describe an autosomal dominant mutation in the SH2 domain of STAT1 that disrupts protein phosphorylation, c.1961T>A (M654K). The mutant allele does not permit STAT1 phosphorylation, and impairs STAT1 phosphorylation of the wild type allele. Protein dimerization is preserved but DNA binding activity, IFN-γ driven GAS-luciferase activity, and expression of IFN-γ target genes are reduced. IFN-α driven ISRE response, but not IFN-α driven GAS response, are preserved when cells are co-transfected with wild type and the mutant STAT1 constructs. M654K exerts a dominant negative effect on IFN-γ related immunity and is recessive for IFN-α induced immune function.
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Infecções por Mycobacterium não Tuberculosas/genética , Complexo Mycobacterium avium/patogenicidade , Fator de Transcrição STAT1/genética , Linfócitos B , Linhagem Celular , Pré-Escolar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Masculino , Mutação , Infecções por Mycobacterium não Tuberculosas/microbiologia , Fosforilação , Multimerização Proteica , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genéticaAssuntos
Mutação com Ganho de Função , Mutação com Perda de Função , Fator de Transcrição STAT3/genética , Substituição de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Síndrome de Job/genética , Transtornos Linfoproliferativos/genética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/imunologia , Eletricidade Estática , Domínios de Homologia de src/genéticaRESUMO
Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.
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COVID-19 , SARS-CoV-2 , Cricetinae , Camundongos , Animais , Humanos , SARS-CoV-2/genética , Mesocricetus , Formação de Anticorpos , Administração Intranasal , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Pulmão/patologia , Camundongos Transgênicos , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , RNA Viral/genética , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Automação , Sensibilidade e EspecificidadeAssuntos
Síndromes de Imunodeficiência/genética , Mucormicose/genética , Fator de Transcrição STAT1/genética , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Interferon gama/farmacologia , Ativação Linfocitária , Masculino , Mucormicose/complicações , Mucormicose/imunologia , Mucormicose/patologia , Mutação , Fosforilação , Fator de Transcrição STAT1/metabolismoRESUMO
Cannabinoid 1 receptor (CB1R) expression is upregulated in the liver with viral hepatitis, cirrhosis, and both alcoholic and non-alcoholic fatty liver disease (FLD), whereas its expression is muted under usual physiological conditions. Inhibiting CB1R has been shown to be beneficial in preserving hepatic function in FLD but it is unclear if inhibiting CB1R during an inflammatory response to an acute hepatic injury, such as toxin-induced injury, would also be beneficial. We found that intrinsic CB1R in hepatocytes regulated liver inflammation-related gene transcription. We tested if nullification of hepatocyte-specific CB1R (hCNR1-/-) in mice protects against concanavalin A (Con A)-induced liver injury. We looked for evidence of liver damage and markers of inflammation in response to Con A by measuring liver enzyme levels and proinflammatory cytokines (e.g., TNF-α, IL-1ß, IL-6, IL-17) in serum collected from hCNR1-/- and control mice. We observed a shift to the right in the dose-response curve for liver injury and inflammation in hCNR1-/- mice. We also found less inflammatory cell infiltration and focal necrosis in livers of hCNR1-/- mice compared to controls, resulting from downregulated apoptotic markers. This anti-apoptotic mechanism results from increased activation of nuclear factor kappa B (NF-κB), especially cAMP-dependent cannabinoid signaling and membrane-bound TNF-α, via downregulated TNF-α receptor 2 (TNFR2) transcription levels. Collectively, these findings provide insight into involvement of CB1R in the pathogenesis of acute liver injury.
Assuntos
Concanavalina A/toxicidade , Hepatócitos/metabolismo , Fígado/metabolismo , Fígado/patologia , NF-kappa B/metabolismo , Receptor CB1 de Canabinoide/deficiência , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Hepatócitos/efeitos dos fármacos , Inflamação/patologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Modelos Biológicos , Ligação Proteica , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In Sub-Saharan Africa, the cancer burden is predicted to increase by > 85% by 2030, the largest increase worldwide. This region has a large HIV-positive population. Drug-drug interactions (DDIs) from concomitant use of multiple drugs increase the risk of drug toxicities, sub-optimal therapy, and drug resistance. With the increase in polypharmacy, involving antiretroviral (ARV), and anticancer drugs, there is a greater need for an appreciation of clinically relevant DDIs. Anticancer and ARV drugs studied in this review were from The World Health Organization's Model List of Essential Medicines 2017. We reviewed; drug package inserts, www.drugbank.ca and www.UpToDate.com, to evaluate pharmacokinetic interactions with cytochrome P450 (CYP450) and ABCB1. The DDIs between drugs were assessed using the University Of Liverpool, UK HIV Drug Interactions Checker, and the LexiComp Drug Interaction tool of www.UpToDate.com. About 70% of ARVs studied interact with CYP450, all involve CYP3A4, and 55% interact with ABCB1. About 65% of anticancer drugs interact with CYP450, 44% of which do so through CYP3A4. About 75% of anticancer drugs interact with ARV drugs, with nine absolute contraindications to concomitant therapy. There exist a substantial number of DDIs between ARV and anticancer drugs, primarily mediated through CYP450 enzymes. Dolutegravir based regimens offer the safest DDI profile for concurrent use with anticancer drugs. However, there are substantial gaps in our knowledge, and this study serves to highlight the need for additional research to better define these interactions and their effect on drug exposure, as attention to these DDIs is a relatively simple intervention that could lead to optimizing disease treatment.