RESUMEN
High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.
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Vacunas contra la COVID-19 , COVID-19 , Humanos , Vacuna BNT162 , COVID-19/prevención & control , Linfocitos T CD8-positivos , Australia/epidemiología , SARS-CoV-2 , Inmunoglobulina G , Anticuerpos Neutralizantes , Inmunidad , Anticuerpos Antivirales , VacunaciónRESUMEN
Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.
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Linfocitos T CD8-positivos/inmunología , Reacciones Cruzadas/inmunología , Gammainfluenzavirus/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Gripe Humana/inmunología , Adolescente , Adulto , Anciano , Animales , Linfocitos T CD8-positivos/virología , Niño , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , Gammainfluenzavirus/fisiología , Masculino , Ratones , Persona de Mediana Edad , Adulto JovenRESUMEN
Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.
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Desmetilasa de ARN, Homólogo 5 de AlkB , Neoplasias de Cabeza y Cuello , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , CarcinogénesisRESUMEN
To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Epítopos Inmunodominantes/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Secuencias de Aminoácidos , Linfocitos T CD4-Positivos , Niño , Convalecencia , Proteínas de la Nucleocápside de Coronavirus/química , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Epítopos Inmunodominantes/química , Masculino , Persona de Mediana Edad , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH). Mechanistically, K413 acetylation of mitochondrial mIDH2 is achieved through a series of hierarchical phosphorylation events mediated by tyrosine kinase FLT3, which phosphorylates mIDH2 to recruit upstream mitochondrial acetyltransferase ACAT1 and simultaneously activates ACAT1 and inhibits upstream mitochondrial deacetylase SIRT3 through tyrosine phosphorylation. Moreover, we found that the intrinsic enzyme activity of mIDH2 is much higher than mIDH1, thus the inhibitory K413 acetylation optimizes leukemogenic ability of mIDH2 in AML cells by both producing sufficient 2-HG for transformation and avoiding cytotoxic accumulation of intracellular 2-HG.
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Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetilación , Animales , Antineoplásicos/farmacología , Femenino , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Lisina/genética , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Mutación/genética , NADP/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Polimorfismo de Nucleótido Simple/genética , Cultivo Primario de Células , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismoRESUMEN
The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.
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COVID-19/patología , COVID-19/virología , Modelos Animales de Enfermedad , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Cricetinae , Femenino , Humanos , Pulmón/patología , Pulmón/virología , Masculino , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Carga ViralRESUMEN
N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.
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Adenina/análogos & derivados , Carcinogénesis/patología , Endopeptidasas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Melanoma/patología , Metiltransferasas/química , Adenina/química , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endopeptidasas/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Metiltransferasas/fisiología , Ratones , Ratones Noqueados , Fosforilación , Estabilidad Proteica , Procesamiento Postranscripcional del ARNRESUMEN
The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.
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Proteínas de Homeodominio , Animales , Ratones , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Neuronas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.
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Adenosina , Metiltransferasas , Ratones Noqueados , Mitocondrias , Animales , Mitocondrias/metabolismo , Mitocondrias/genética , Ratones , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , ARN/genética , ARN/metabolismo , Humanos , Biosíntesis de Proteínas , Metabolismo Energético/genética , Neuronas/metabolismo , Metilación de ARNRESUMEN
BACKGROUND: The addition of vancomycin to beta-lactam prophylaxis in arthroplasty may reduce surgical-site infections; however, the efficacy and safety are unclear. METHODS: In this multicenter, double-blind, superiority, placebo-controlled trial, we randomly assigned adult patients without known methicillin-resistant Staphylococcus aureus (MRSA) colonization who were undergoing arthroplasty to receive 1.5 g of vancomycin or normal saline placebo, in addition to cefazolin prophylaxis. The primary outcome was surgical-site infection within 90 days after surgery. RESULTS: A total of 4239 patients underwent randomization. Among 4113 patients in the modified intention-to-treat population (2233 undergoing knee arthroplasty, 1850 undergoing hip arthroplasty, and 30 undergoing shoulder arthroplasty), surgical-site infections occurred in 91 of 2044 patients (4.5%) in the vancomycin group and in 72 of 2069 patients (3.5%) in the placebo group (relative risk, 1.28; 95% confidence interval [CI], 0.94 to 1.73; P = 0.11). Among patients undergoing knee arthroplasty, surgical-site infections occurred in 63 of 1109 patients (5.7%) in the vancomyin group and in 42 of 1124 patients (3.7%) in the placebo group (relative risk, 1.52; 95% CI, 1.04 to 2.23). Among patients undergoing hip arthroplasty, surgical-site infections occurred in 28 of 920 patients (3.0%) in the vancomyin group and in 29 of 930 patients (3.1%) in the placebo group (relative risk, 0.98; 95% CI, 0.59 to 1.63). Adverse events occurred in 35 of 2010 patients (1.7%) in the vancomycin group and in 35 of 2030 patients (1.7%) in the placebo group, including hypersensitivity reactions in 24 of 2010 patients (1.2%) and 11 of 2030 patients (0.5%), respectively (relative risk, 2.20; 95% CI, 1.08 to 4.49), and acute kidney injury in 42 of 2010 patients (2.1%) and 74 of 2030 patients (3.6%), respectively (relative risk, 0.57; 95% CI, 0.39 to 0.83). CONCLUSIONS: The addition of vancomycin to cefazolin prophylaxis was not superior to placebo for the prevention of surgical-site infections in arthroplasty among patients without known MRSA colonization. (Funded by the Australian National Health and Medical Research Council; Australian New Zealand Clinical Trials Registry number, ACTRN12618000642280.).
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Antibacterianos , Profilaxis Antibiótica , Artroplastia de Reemplazo , Cefazolina , Infección de la Herida Quirúrgica , Vancomicina , Adulto , Humanos , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Profilaxis Antibiótica/efectos adversos , Profilaxis Antibiótica/métodos , Artroplastia de Reemplazo de Rodilla/efectos adversos , Artroplastia de Reemplazo de Rodilla/métodos , Australia , Cefazolina/efectos adversos , Cefazolina/uso terapéutico , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/tratamiento farmacológico , Infección de la Herida Quirúrgica/epidemiología , Infección de la Herida Quirúrgica/prevención & control , Infección de la Herida Quirúrgica/etiología , Vancomicina/efectos adversos , Vancomicina/uso terapéutico , Método Doble Ciego , Artroplastia de Reemplazo/efectos adversos , Artroplastia de Reemplazo/métodos , Artroplastia de Reemplazo/estadística & datos numéricosRESUMEN
BACKGROUND: The efficacy of simvastatin in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. METHODS: In an ongoing international, multifactorial, adaptive platform, randomized, controlled trial, we evaluated simvastatin (80 mg daily) as compared with no statin (control) in critically ill patients with Covid-19 who were not receiving statins at baseline. The primary outcome was respiratory and cardiovascular organ support-free days, assessed on an ordinal scale combining in-hospital death (assigned a value of -1) and days free of organ support through day 21 in survivors; the analyis used a Bayesian hierarchical ordinal model. The adaptive design included prespecified statistical stopping criteria for superiority (>99% posterior probability that the odds ratio was >1) and futility (>95% posterior probability that the odds ratio was <1.2). RESULTS: Enrollment began on October 28, 2020. On January 8, 2023, enrollment was closed on the basis of a low anticipated likelihood that prespecified stopping criteria would be met as Covid-19 cases decreased. The final analysis included 2684 critically ill patients. The median number of organ support-free days was 11 (interquartile range, -1 to 17) in the simvastatin group and 7 (interquartile range, -1 to 16) in the control group; the posterior median adjusted odds ratio was 1.15 (95% credible interval, 0.98 to 1.34) for simvastatin as compared with control, yielding a 95.9% posterior probability of superiority. At 90 days, the hazard ratio for survival was 1.12 (95% credible interval, 0.95 to 1.32), yielding a 91.9% posterior probability of superiority of simvastatin. The results of secondary analyses were consistent with those of the primary analysis. Serious adverse events, such as elevated levels of liver enzymes and creatine kinase, were reported more frequently with simvastatin than with control. CONCLUSIONS: Although recruitment was stopped because cases had decreased, among critically ill patients with Covid-19, simvastatin did not meet the prespecified criteria for superiority to control. (REMAP-CAP ClinicalTrials.gov number, NCT02735707.).
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COVID-19 , Enfermedad Crítica , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Simvastatina , Humanos , Teorema de Bayes , COVID-19/mortalidad , COVID-19/terapia , Tratamiento Farmacológico de COVID-19 , Enfermedad Crítica/mortalidad , Enfermedad Crítica/terapia , Mortalidad Hospitalaria , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Simvastatina/uso terapéutico , Resultado del TratamientoRESUMEN
The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell-cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM.
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Cardiomiopatía Dilatada , Insuficiencia Cardíaca , Animales , Ratones , Humanos , Cardiomiopatía Dilatada/metabolismo , Ventrículos Cardíacos/metabolismo , Fosforilación , Proteómica , Miocardio/metabolismo , Insuficiencia Cardíaca/metabolismo , alfa Catenina/metabolismoRESUMEN
BACKGROUND: Frequent anti-vascular endothelial growth factor A (VEGF-A) injections reduce the risk of rapid and severe vision loss in patients with neovascular age-related macular degeneration (nAMD); however, due to undertreatment, many patients lose vision over time. New treatments that provide sustained suppression of VEGF-A are needed. RGX-314 (currently known as ABBV-RGX-314) is an adeno-associated virus serotype 8 vector that expresses an anti-VEGF-A antigen-binding fragment, which provides potential for continuous VEGF-A suppression after a single subretinal injection. We report results on the safety and efficacy of subretinal injection of RGX-314 in patients with nAMD. METHODS: For this open-label, multiple-cohort, multicentre, phase 1/2a, dose-escalation study conducted at eight sites in the USA, we enrolled participants with nAMD aged 50-89 years who had previously been treated with anti-VEGF injections into five cohorts (with five different doses of RGX-314). To be eligible, participants had to have macular neovascularisation secondary to nAMD with subretinal or intraretinal fluid in the centre subfield, be pseudophakic (after cataract removal), and have a best-corrected visual acuity (BCVA) in the study eye between 20/63 and 20/400 for the first participant in each cohort and between 20/40 and 20/400 for others. Subretinal injection of RGX-314 was done without a pre-bleb by a wet-laboratory-trained vitreoretinal surgeon. Cohort 1 received 3 × 109 genome copies per eye, cohort 2 received 1 × 1010, and cohort 3 received 6 × 1010. Two additional dose cohorts (cohort 4: 1·6 × 1011; cohort 5: 2·5 × 1011) were added. Participants were seen 1 day and 1 week after administration of RGX-314, and then monthly for 2 years (up to week 106). The primary outcome was safety of RGX-314 delivered by subretinal injection up to week 26. This analysis includes all 42 patients enrolled in the study. This study is registered with ClinicalTrials.gov, NCT03066258. FINDINGS: Between May 12, 2017, and May 21, 2019, we screened 110 patients for eligibility and enrolled 68. 42 participants demonstrated the required anatomic response to intravitreal ranibizumab and then received a single RGX-314 injection (dose range 3 × 109 to 2·5 × 1011 genome copies per eye) and were followed up for 2 years. There were 20 serious adverse events in 13 participants, of which one was possibly related to RGX-314: pigmentary changes in the macula with severe vision reduction 12 months after injection of RGX-314 at a dose of 2·5 × 1011 genome copies per eye. Asymptomatic pigmentary changes were seen in the inferior retinal periphery several months after subretinal injection of RGX-314 most commonly at doses of 6 × 1010 genome copies per eye or higher. There were no clinically determined immune responses or inflammation beyond that expected following routine vitrectomy. Doses of 6 × 1010 genome copies or higher resulted in sustained concentrations of RGX-314 protein in aqueous humour and stable or improved BCVA and central retinal thickness with few or no supplemental anti-VEGF-A injections in most participants. INTERPRETATION: Subretinal delivery of RGX-314 was generally well tolerated with no clinically recognised immune responses. RGX-314 gene therapy provides a novel approach for sustained VEGF-A suppression in patients with nAMD that has potential to control exudation, maintain vision, and reduce treatment burden after a single administration. Results from this study informed the pivotal programme to evaluate RGX-314 in patients with nAMD. FUNDING: RegenxBio.
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Factor A de Crecimiento Endotelial Vascular , Degeneración Macular Húmeda , Humanos , Inhibidores de la Angiogénesis/uso terapéutico , Terapia Genética/métodos , Ranibizumab , Resultado del Tratamiento , Degeneración Macular Húmeda/tratamiento farmacológicoRESUMEN
Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.
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Borrelia burgdorferi , Enfermedad de Lyme , Humanos , Borrelia burgdorferi/genética , Genotipo , Secuenciación Completa del Genoma , Plásmidos/genéticaRESUMEN
Lyme arthritis (LA) was recognized as a separate entity in 1975 because of geographic clustering of children often diagnosed with juvenile rheumatoid arthritis in Lyme, Connecticut. After identification of erythema migrans as a common early feature of the illness, a prospective study of such patients implicated Ixodes scapularis ticks in disease transmission. In 1982, the causative agent, now called Borrelia burgdorferi, was cultured from these ticks and from Lyme disease patients. Subsequently, it was shown that LA could usually be treated successfully with oral antibiotics but sometimes required intravenous antibiotics. Yet, a small percentage of patients developed a dysregulated, proinflammatory immune response leading to persistent postinfectious synovitis with vascular damage, cytotoxic and autoimmune responses, and fibroblast proliferation, a lesion similar to that of rheumatoid arthritis. The message from postinfectious LA for other autoimmune arthritides is that a complex immune response with autoimmune features can begin with a microbial infection.
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Enfermedad de Lyme , Enfermedad de Lyme/inmunología , Humanos , Animales , Historia del Siglo XX , Borrelia burgdorferi/inmunología , Historia del Siglo XXI , Antibacterianos/uso terapéutico , Ixodes/microbiologíaRESUMEN
BACKGROUND: Postinfectious Lyme arthritis (LA) is associated with dysregulated immunity and autoreactive T- and B-cell responses in joints. Here we explored the role of host genetic variation in this outcome. METHODS: The frequency of 253 702 single-nucleotide polymorphisms (SNPs) was determined in 147 patients with LA (87 with postinfectious LA and 60 with antibiotic-responsive LA), and for comparison in 90 patients with erythema migrans or the general population (n = 2504). Functional outcome of candidate SNPs was assessed by evaluating their impact on clinical outcome and on immune responses in blood and synovial fluid in patients with LA. RESULTS: Six SNPs associated with late cornified envelope (LCE3) genes were present at greater frequency in patients with postinfectious LA compared to those with antibiotic-responsive LA (70% vs 30%; odds ratio, 2; P < .01). These SNPs were associated with heightened levels of inflammatory Th17 cytokines in serum but lower levels of interleukin 27, a regulatory cytokine, implying that they may contribute to dysregulated Th17 immunity in blood. Moreover, in patients with postinfectious LA, the levels of these Th17 mediators correlated directly with autoantibody responses in synovial fluid, providing a possible link between LCE3 SNPs, maladaptive systemic Th17 immunity, and autoreactive responses in joints. CONCLUSIONS: Variation in the LCE3 locus, a known genetic risk factor in psoriasis and psoriatic arthritis, is associated with dysregulated systemic Th17 immunity and heightened autoantibody responses in joints. These findings underscore the importance of host genetic predisposition and systemic Th17 immunity in the pathogenesis of postinfectious (antibiotic-refractory) Lyme arthritis.
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Enfermedad de Lyme , Polimorfismo de Nucleótido Simple , Células Th17 , Humanos , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Células Th17/inmunología , Masculino , Femenino , Adulto , Persona de Mediana Edad , Líquido Sinovial/inmunología , Anciano , Citocinas/genética , Citocinas/metabolismo , Artritis Infecciosa/genética , Artritis Infecciosa/inmunología , Adulto JovenRESUMEN
Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.
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Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Proteína p53 Supresora de Tumor , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Resonancia Magnética Nuclear Biomolecular , Espectroscopía de Resonancia MagnéticaRESUMEN
BACKGROUND: The glucocorticoid dexamethasone prevents nausea and vomiting after surgery, but there is concern that it may increase the risk of surgical-site infection. METHODS: In this pragmatic, international, noninferiority trial, we randomly assigned 8880 adult patients who were undergoing nonurgent, noncardiac surgery of at least 2 hours' duration, with a skin incision length longer than 5 cm and a postoperative overnight hospital stay, to receive 8 mg of intravenous dexamethasone or matching placebo while under anesthesia. Randomization was stratified according to diabetes status and trial center. The primary outcome was surgical-site infection within 30 days after surgery. The prespecified noninferiority margin was 2.0 percentage points. RESULTS: A total of 8725 participants were included in the modified intention-to-treat population (4372 in the dexamethasone group and 4353 in the placebo group), of whom 13.2% (576 in the dexamethasone group and 572 in the placebo group) had diabetes mellitus. Of the 8678 patients included in the primary analysis, surgical-site infection occurred in 8.1% (354 of 4350 patients) assigned to dexamethasone and in 9.1% (394 of 4328) assigned to placebo (risk difference adjusted for diabetes status, -0.9 percentage points; 95.6% confidence interval [CI], -2.1 to 0.3; P<0.001 for noninferiority). The results for superficial, deep, and organ-space surgical-site infections and in patients with diabetes were similar to those of the primary analysis. Postoperative nausea and vomiting in the first 24 hours after surgery occurred in 42.2% of patients in the dexamethasone group and in 53.9% in the placebo group (risk ratio, 0.78; 95% CI, 0.75 to 0.82). Hyperglycemic events in patients without diabetes occurred in 22 of 3787 (0.6%) in the dexamethasone group and in 6 of 3776 (0.2%) in the placebo group. CONCLUSIONS: Dexamethasone was noninferior to placebo with respect to the incidence of surgical-site infection within 30 days after nonurgent, noncardiac surgery. (Funded by the Australian National Health and Medical Research Council and others; PADDI Australian New Zealand Clinical Trials Registry number, ACTRN12614001226695.).
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Antieméticos/efectos adversos , Dexametasona/efectos adversos , Glucocorticoides/efectos adversos , Procedimientos Quirúrgicos Operativos/efectos adversos , Infección de la Herida Quirúrgica/etiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anestesia General , Antieméticos/uso terapéutico , Dexametasona/uso terapéutico , Método Doble Ciego , Femenino , Glucocorticoides/uso terapéutico , Humanos , Análisis de Intención de Tratar , Masculino , Persona de Mediana Edad , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: Thrombosis and inflammation may contribute to morbidity and mortality among patients with coronavirus disease 2019 (Covid-19). We hypothesized that therapeutic-dose anticoagulation would improve outcomes in critically ill patients with Covid-19. METHODS: In an open-label, adaptive, multiplatform, randomized clinical trial, critically ill patients with severe Covid-19 were randomly assigned to a pragmatically defined regimen of either therapeutic-dose anticoagulation with heparin or pharmacologic thromboprophylaxis in accordance with local usual care. The primary outcome was organ support-free days, evaluated on an ordinal scale that combined in-hospital death (assigned a value of -1) and the number of days free of cardiovascular or respiratory organ support up to day 21 among patients who survived to hospital discharge. RESULTS: The trial was stopped when the prespecified criterion for futility was met for therapeutic-dose anticoagulation. Data on the primary outcome were available for 1098 patients (534 assigned to therapeutic-dose anticoagulation and 564 assigned to usual-care thromboprophylaxis). The median value for organ support-free days was 1 (interquartile range, -1 to 16) among the patients assigned to therapeutic-dose anticoagulation and was 4 (interquartile range, -1 to 16) among the patients assigned to usual-care thromboprophylaxis (adjusted proportional odds ratio, 0.83; 95% credible interval, 0.67 to 1.03; posterior probability of futility [defined as an odds ratio <1.2], 99.9%). The percentage of patients who survived to hospital discharge was similar in the two groups (62.7% and 64.5%, respectively; adjusted odds ratio, 0.84; 95% credible interval, 0.64 to 1.11). Major bleeding occurred in 3.8% of the patients assigned to therapeutic-dose anticoagulation and in 2.3% of those assigned to usual-care pharmacologic thromboprophylaxis. CONCLUSIONS: In critically ill patients with Covid-19, an initial strategy of therapeutic-dose anticoagulation with heparin did not result in a greater probability of survival to hospital discharge or a greater number of days free of cardiovascular or respiratory organ support than did usual-care pharmacologic thromboprophylaxis. (REMAP-CAP, ACTIV-4a, and ATTACC ClinicalTrials.gov numbers, NCT02735707, NCT04505774, NCT04359277, and NCT04372589.).
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Anticoagulantes/administración & dosificación , Tratamiento Farmacológico de COVID-19 , Heparina/administración & dosificación , Trombosis/prevención & control , Anciano , Anticoagulantes/efectos adversos , Anticoagulantes/uso terapéutico , COVID-19/mortalidad , Enfermedad Crítica , Femenino , Hemorragia/inducido químicamente , Heparina/efectos adversos , Heparina/uso terapéutico , Mortalidad Hospitalaria , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Respiración Artificial , Insuficiencia del TratamientoRESUMEN
BACKGROUND: Thrombosis and inflammation may contribute to the risk of death and complications among patients with coronavirus disease 2019 (Covid-19). We hypothesized that therapeutic-dose anticoagulation may improve outcomes in noncritically ill patients who are hospitalized with Covid-19. METHODS: In this open-label, adaptive, multiplatform, controlled trial, we randomly assigned patients who were hospitalized with Covid-19 and who were not critically ill (which was defined as an absence of critical care-level organ support at enrollment) to receive pragmatically defined regimens of either therapeutic-dose anticoagulation with heparin or usual-care pharmacologic thromboprophylaxis. The primary outcome was organ support-free days, evaluated on an ordinal scale that combined in-hospital death (assigned a value of -1) and the number of days free of cardiovascular or respiratory organ support up to day 21 among patients who survived to hospital discharge. This outcome was evaluated with the use of a Bayesian statistical model for all patients and according to the baseline d-dimer level. RESULTS: The trial was stopped when prespecified criteria for the superiority of therapeutic-dose anticoagulation were met. Among 2219 patients in the final analysis, the probability that therapeutic-dose anticoagulation increased organ support-free days as compared with usual-care thromboprophylaxis was 98.6% (adjusted odds ratio, 1.27; 95% credible interval, 1.03 to 1.58). The adjusted absolute between-group difference in survival until hospital discharge without organ support favoring therapeutic-dose anticoagulation was 4.0 percentage points (95% credible interval, 0.5 to 7.2). The final probability of the superiority of therapeutic-dose anticoagulation over usual-care thromboprophylaxis was 97.3% in the high d-dimer cohort, 92.9% in the low d-dimer cohort, and 97.3% in the unknown d-dimer cohort. Major bleeding occurred in 1.9% of the patients receiving therapeutic-dose anticoagulation and in 0.9% of those receiving thromboprophylaxis. CONCLUSIONS: In noncritically ill patients with Covid-19, an initial strategy of therapeutic-dose anticoagulation with heparin increased the probability of survival to hospital discharge with reduced use of cardiovascular or respiratory organ support as compared with usual-care thromboprophylaxis. (ATTACC, ACTIV-4a, and REMAP-CAP ClinicalTrials.gov numbers, NCT04372589, NCT04505774, NCT04359277, and NCT02735707.).