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The isolation of viruses from complex biological samples is essential for creating sensitive bioassays that assess the efficacy and safety of viral therapeutics and vaccines, which have played a critical role during the COVID-19 pandemic. However, existing methods of viral isolation are time-consuming and labor-intensive due to the multiple processing steps required, resulting in low yields. Here, we introduce the rapid, efficient, and high-resolution acoustofluidic isolation of viruses from complex biological samples via Bessel beam excitation separation technology (BEST). BEST isolates viruses by utilizing the nondiffractive and self-healing properties of 2D, in-plane acoustic Bessel beams to continuously separate cell-free viruses from biofluids, with high throughput and high viral RNA yield. By tuning the acoustic parameters, the cutoff size of isolated viruses can be easily adjusted to perform dynamic, size-selective virus isolation while simultaneously trapping larger particles and separating smaller particles and contaminants from the sample, achieving high-precision isolation of the target virus. BEST was used to isolate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human saliva samples and Moloney Murine Leukemia Virus from cell culture media, demonstrating its potential use in both practical diagnostic applications and fundamental virology research. With high separation resolution, high yield, and high purity, BEST is a powerful tool for rapidly and efficiently isolating viruses. It has the potential to play an important role in the development of next-generation viral diagnostics, therapeutics, and vaccines.
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Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.
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Separación Celular , Células Endoteliales , Citometría de Flujo , Pulmón , Animales , Ratones , Células Endoteliales/citología , Células Endoteliales/metabolismo , Pulmón/citología , Separación Celular/métodos , Citometría de Flujo/métodos , Colagenasas/metabolismo , Análisis de la Célula Individual/métodos , Ratones Endogámicos C57BL , EndopeptidasasRESUMEN
BACKGROUND: Despite causing increased morbidity and mortality, pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) patients (COPD-PH) lacks treatment, due to incomplete understanding of its pathogenesis. Hypertrophy of pulmonary arterial walls and pruning of the microvasculature with loss of capillary beds are known features of pulmonary vascular remodeling in COPD. The remodeling features of pulmonary medium- and smaller vessels in COPD-PH lungs are less well described and may be linked to maladaptation of endothelial cells to chronic cigarette smoking (CS). MicroRNA-126 (miR126), a master regulator of endothelial cell fate, has divergent functions that are vessel-size specific, supporting the survival of large vessel endothelial cells and inhibiting the proliferation of microvascular endothelial cells. Since CS decreases miR126 in microvascular lung endothelial cells, we set out to characterize the remodeling by pulmonary vascular size in COPD-PH and its relationship with miR126 in COPD and COPD-PH lungs. METHODS: Deidentified lung tissue was obtained from individuals with COPD with and without PH and from non-diseased non-smokers and smokers. Pulmonary artery remodeling was assessed by âº-smooth muscle actin (SMA) abundance via immunohistochemistry and analyzed by pulmonary artery size. miR126 and miR126-target abundance were quantified by qPCR. The expression levels of ceramide, ADAM9, and endothelial cell marker CD31 were assessed by immunofluorescence. RESULTS: Pulmonary arteries from COPD and COPD-PH lungs had significantly increased SMA abundance compared to non-COPD lungs, especially in small pulmonary arteries and the lung microvasculature. This was accompanied by significantly fewer endothelial cell markers and increased pro-apoptotic ceramide abundance. miR126 expression was significantly decreased in lungs of COPD individuals. Of the targets tested (SPRED1, VEGF, LAT1, ADAM9), lung miR126 most significantly inversely correlated with ADAM9 expression. Compared to controls, ADAM9 was significantly increased in COPD and COPD-PH lungs, predominantly in small pulmonary arteries and lung microvasculature. CONCLUSION: Both COPD and COPD-PH lungs exhibited significant remodeling of the pulmonary vascular bed of small and microvascular size, suggesting these changes may occur before or independent of the clinical development of PH. Decreased miR126 expression with reciprocal increase in ADAM9 may regulate endothelial cell survival and vascular remodeling in small pulmonary arteries and lung microvasculature in COPD and COPD-PH.
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Hipertensión Pulmonar , MicroARNs , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Hipertensión Pulmonar/patología , Remodelación Vascular , Células Endoteliales/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Arteria Pulmonar/metabolismo , Pulmón/metabolismo , Ceramidas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/metabolismoRESUMEN
Epithelial cells of diverse tissues are characterized by the presence of a single apical domain. In the lung, electron microscopy studies have suggested that alveolar type-2 epithelial cells (AT2s) en face multiple alveolar sacs. However, apical and basolateral organization of the AT2s and their establishment during development and remodeling after injury repair remain unknown. Thick tissue imaging and electron microscopy revealed that a single AT2 can have multiple apical domains that enface multiple alveoli. AT2s gradually establish multi-apical domains post-natally, and they are maintained throughout life. Lineage tracing, live imaging, and selective cell ablation revealed that AT2s dynamically reorganize multi-apical domains during injury repair. Single-cell transcriptome signatures of residual AT2s revealed changes in cytoskeleton and cell migration. Significantly, cigarette smoke and oncogene activation lead to dysregulation of multi-apical domains. We propose that the multi-apical domains of AT2s enable them to be poised to support the regeneration of a large array of alveolar sacs.
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Importance: Health care workers (HCWs) have been experiencing substantial stress and burnout, and evidence-based mitigation strategies are needed. Transcendental Meditation (TM) is a mantra meditation practice with potential efficacy in reducing stress. Objective: To assess the efficacy of TM practice in reducing stress among HCWs over a 3-month period. Design, Setting, and Participants: This single-center open-label randomized clinical trial was conducted among HCWs at an academic medical center from November 19, 2020, to August 31, 2021. Inclusion criteria comprised a score of 6 points or greater on the Subjective Units of Distress Scale and an increase of 5% or greater in baseline heart rate or an increase of 33% or greater in galvanic skin response after exposure to a stressful script. Exclusion criteria included the use of antipsychotic or ß blocker medications, current suicidal ideation, or previous TM training. Of 213 HCWs who participated in prescreening, 95 attended in-person visits, resulting in 80 eligible participants who were randomized to receive a TM intervention (TM group) or usual treatment (control group). Interventions: The TM group practiced TM for 20 minutes twice daily over a 3-month period. The control group received usual treatment, which consisted of access to wellness resources. Main Outcomes and Measures: The primary outcome was change in acute psychological distress measured by the Global Severity Index. Secondary outcomes included changes in burnout (measured by the Maslach Burnout Inventory), insomnia (measured by the Insomnia Severity Index), and anxiety (measured by the Generalized Anxiety Disorder-7 scale). Results: Among 80 participants, 66 (82.5%) were women, with a mean (SD) age of 40 (11) years. One participant (1.3%) was American Indian or Alaska Native, 5 (6.3%) were Asian, 12 (15.0%) were Black, 59 (73.8%) were White, and 3 (3.8%) were of unknown or unreported race; 4 participants (5.0%) were Hispanic, and 76 (95.0%) were non-Hispanic. A total of 41 participants were randomized to the TM group, and 39 were randomized to the control group. Participants in the TM group did not show a statistically significant decrease in psychological distress on the Global Severity Index compared with those in the control group (-5.6 points vs -3.8 points; between-group difference, -1.8 points; 95% CI, -4.2 to 0.6 points; P = .13). Compared with the control group, the TM group had significantly greater reductions in the secondary end points of emotional exhaustion (Maslach Burnout Inventory subscore: -8.0 points vs -2.6 points; between-group difference, -5.4 points; 95% CI, -9.2 to -1.6 points; P = .006), insomnia (Insomnia Severity Scale score: -4.1 points vs -1.9 points; between-group difference, -2.2 points; 95% CI, -4.4 to 0 points; P = .05), and anxiety (Generalized Anxiety Disorder-7 score: -3.1 points vs -0.9 points; between-group difference, -2.2 points; 95% CI, -3.8 to -0.5; P = .01) at 3 months. A total of 38 participants (92.7%) in the TM group adhered to home practice. Conclusions and Relevance: In this randomized clinical trial, TM practice among HCWs over a 3-month period did not result in a statistically significant reduction in the primary outcome of acute psychological distress compared with usual treatment but significantly improved the secondary outcomes of burnout, anxiety, and insomnia. These findings suggest that TM may be a safe and effective strategy to alleviate chronic stress among HCWs. Trial Registration: ClinicalTrials.gov identifier: NCT04632368.
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Antipsicóticos , Agotamiento Profesional , Meditación , Trastornos del Inicio y del Mantenimiento del Sueño , Adulto , Agotamiento Profesional/prevención & control , Femenino , Personal de Salud , Humanos , MasculinoRESUMEN
Pulmonary hypertension (PH) is a debilitating condition characterized by increased pulmonary arterial pressures and remodeling of pulmonary arteries, leading to right heart failure. Women have a higher prevalence of PH, whereas men have more severe disease and poorer outcomes. Animal models also show female-predominant disease. Despite the known sex differences in PH, little is known about how pathogenesis differs between the sexes. There is growing evidence of mitochondrial dysfunction, as well as altered mitophagy in PH. We hypothesized that sexual dimorphism contributes to mitochondrial dysfunction and altered mitophagy in PH. Using mouse lung endothelial cells, we exposed both wild-type and Parkin-/- cells to hypoxia and measured the effects on mitochondrial function and mitophagy-associated proteins. Our results show that females have more Parkin expression at baseline as well as increased mitochondrial respiratory capacity when exposed to oxidative stress. Inhibition of Parkin increased metabolic activity but reduced cell proliferation but to different degrees depending on sex, with results differing by sex. Our findings demonstrate sexual dimorphism in mitophagy-associated proteins and in mitochondrial respiration, which may help shed light on how the pathogenesis of PH may differ between the sexes.
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Hipertensión Pulmonar , Mitofagia , Animales , Células Endoteliales/metabolismo , Femenino , Humanos , Hipertensión Pulmonar/metabolismo , Masculino , Ratones , Mitofagia/fisiología , Caracteres Sexuales , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUNDIndividuals recovering from COVID-19 frequently experience persistent respiratory ailments, which are key elements of postacute sequelae of SARS-CoV-2 infection (PASC); however, little is known about the underlying biological factors that may direct lung recovery and the extent to which these are affected by COVID-19 severity.METHODSWe performed a prospective cohort study of individuals with persistent symptoms after acute COVID-19, collecting clinical data, pulmonary function tests, and plasma samples used for multiplex profiling of inflammatory, metabolic, angiogenic, and fibrotic factors.RESULTSSixty-one participants were enrolled across 2 academic medical centers at a median of 9 weeks (interquartile range, 6-10 weeks) after COVID-19 illness: n = 13 participants (21%) had mild COVID-19 and were not hospitalized, n = 30 participants (49%) were hospitalized but were considered noncritical, and n = 18 participants (30%) were hospitalized and in the intensive care unit (ICU). Fifty-three participants (85%) had lingering symptoms, most commonly dyspnea (69%) and cough (58%). Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and diffusing capacity for carbon monoxide (DLCO) declined as COVID-19 severity increased (P < 0.05) but these values did not correlate with respiratory symptoms. Partial least-squares discriminant analysis of plasma biomarker profiles clustered participants by past COVID-19 severity. Lipocalin-2 (LCN2), MMP-7, and HGF identified by our analysis were significantly higher in the ICU group (P < 0.05), inversely correlated with FVC and DLCO (P < 0.05), and were confirmed in a separate validation cohort (n = 53).CONCLUSIONSubjective respiratory symptoms are common after acute COVID-19 illness but do not correlate with COVID-19 severity or pulmonary function. Host response profiles reflecting neutrophil activation (LCN2), fibrosis signaling (MMP-7), and alveolar repair (HGF) track with lung impairment and may be novel therapeutic or prognostic targets.FundingNational Heart, Lung, and Blood Institute (K08HL130557 and R01HL142818), American Heart Association (Transformational Project Award), the DeLuca Foundation Award, a donation from Jack Levin to the Benign Hematology Program at Yale University, and Duke University.
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COVID-19/complicaciones , Factor de Crecimiento de Hepatocito/análisis , Lipocalina 2/análisis , Metaloproteinasa 7 de la Matriz/análisis , Fibrosis Pulmonar , Pruebas de Función Respiratoria , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/fisiopatología , Tos/diagnóstico , Tos/etiología , Disnea/diagnóstico , Disnea/etiología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Activación Neutrófila/inmunología , Pronóstico , Fibrosis Pulmonar/diagnóstico , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismo , Recuperación de la Función/inmunología , Pruebas de Función Respiratoria/métodos , Pruebas de Función Respiratoria/estadística & datos numéricos , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Síndrome Post Agudo de COVID-19RESUMEN
Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47phox Ser208 to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47phox pathway acted via paracrine H2O2 to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.
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Lesión Pulmonar Aguda , Indazoles/farmacología , MAP Quinasa Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 3/antagonistas & inhibidores , Pirimidinas/farmacología , Sulfonamidas/farmacología , Lesión Pulmonar Aguda/tratamiento farmacológico , Animales , Humanos , Peróxido de Hidrógeno , Ratones , NADPH Oxidasas/metabolismo , Fosforilación , Especies Reactivas de OxígenoRESUMEN
Hyperoxia can lead to respiratory failure and death. Our previous work demonstrates that oxidant and mitochondrial injury play a critical role in hyperoxia-induced acute lung injury (HALI). Recently, thyroid hormone has been demonstrated to promote mitochondrial survival in other models of lung injury, but its role in hyperoxia is unknown. Adult wild-type (WT) mice were pretreated with either nebulized triiodothyronine (T3, 40 µg/kg) for 1 or 3 days, or with propylthiouracil (PTU, 100 µg/kg), for 3 days. Following pretreatment, WT mice underwent 72 h of hyperoxia exposure. WT and PINK1-/- mice were pretreated with either nebulized T3 (40 µg/kg) for 3 days or no pretreatment before 72 h continuous hyperoxia exposure. Bronchoalveolar lavage (BAL), histological changes in cellular composition, and type I cytokine induction were assessed. Lung lysates for mitochondrial cellular bioenergetics markers were analyzed by Western blot. Hyperoxia caused a significant increase in BAL total cell counts and lung cellular infiltrates. Administration of PTU enhanced HALI, whereas T3 attenuated HALI, inflammation, and oxidants in WT mice. In addition, T3 pretreatment increased mitochondrial biogenesis/fusion/mitophagy and decreased ER stress and apoptosis. PINK1-/- mice were more susceptible to hyperoxia than WT mice. Notably, pretreatment with T3 did not attenuate HALI in PINK1-/- mice. In addition, T3 pretreatment increased mitochondrial anti-ROS potential, improved mitochondrial bioenergetics and mitophagy, and attenuated mitochondria-regulated apoptosis, all in a PINK1-dependent manner. Our results highlight a novel protective role for PINK1 in mediating the cytoprotective effects of thyroid hormone in HALI. Therefore, thyroid hormone may represent a potential therapy for ALI.
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Hiperoxia/patología , Lesión Pulmonar/patología , Proteínas Quinasas/metabolismo , Triyodotironina/farmacología , Animales , Inflamación/tratamiento farmacológico , Inflamación/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Triyodotironina/metabolismoRESUMEN
INTRODUCTION: Subjects recovering from COVID-19 frequently experience persistent respiratory ailments; however, little is known about the underlying biological factors that may direct lung recovery and the extent to which these are affected by COVID-19 severity. METHODS: We performed a prospective cohort study of subjects with persistent symptoms after acute COVID-19, collecting clinical data, pulmonary function tests, and plasma samples used for multiplex profiling of inflammatory, metabolic, angiogenic, and fibrotic factors. RESULTS: Sixty-one subjects were enrolled across two academic medical centers at a median of 9 weeks (interquartile range 6-10) after COVID-19 illness: n=13 subjects (21%) mild/non-hospitalized, n=30 (49%) hospitalized/non-critical, and n=18 subjects (30%) hospitalized/intensive care ("ICU"). Fifty-three subjects (85%) had lingering symptoms, most commonly dyspnea (69%) and cough (58%). Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and diffusing capacity for carbon monoxide (DLCO) declined as COVID-19 severity increased (P<0.05), but did not correlate with respiratory symptoms. Partial least-squares discriminant analysis of plasma biomarker profiles clustered subjects by past COVID-19 severity. Lipocalin 2 (LCN2), matrix metalloproteinase-7 (MMP-7), and hepatocyte growth factor (HGF) identified by the model were significantly higher in the ICU group (P<0.05) and inversely correlated with FVC and DLCO (P<0.05), and were confirmed in a separate validation cohort (n=53). CONCLUSIONS: Subjective respiratory symptoms are common after acute COVID-19 illness but do not correlate with COVID-19 severity or pulmonary function. Host response profiles reflecting neutrophil activation (LCN2), fibrosis signaling (MMP-7), and alveolar repair (HGF) track with lung impairment and may be novel therapeutic or prognostic targets. FUNDING: The study was funded in part by the NHLBI (K08HL130557 to BDK and R01HL142818 to HJC), the DeLuca Foundation Award (AP), a donation from Jack Levin to the Benign Hematology Program at Yale, and Divisional/Departmental funds from Duke University.
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The pathogenesis of chronic obstructive pulmonary disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema and validated this finding in multiple cohorts. In a CS exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 were increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlated with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.
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Apoptosis/genética , Daño del ADN/genética , MicroARNs/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular , Fumar Cigarrillos/efectos adversos , Estudios de Cohortes , Reparación del ADN , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos AKR , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here, we report a feeder-free, scalable, chemically defined, and modular alveolosphere culture system for the propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor angiotensin-converting enzyme receptor type-2 (ACE2) and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirror features of COVID-19 lungs, including emergence of interferon (IFN)-mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with IFNs recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low-dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres, thus, provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases.
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Células Madre Adultas/virología , Células Epiteliales Alveolares/efectos de los fármacos , Tratamiento Farmacológico de COVID-19 , Interferones/farmacología , SARS-CoV-2/inmunología , Adulto , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/enzimología , Anciano , Anciano de 80 o más Años , Células Epiteliales Alveolares/enzimología , Células Epiteliales Alveolares/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/fisiopatología , Técnicas de Cultivo de Célula , Diferenciación Celular , Femenino , Humanos , Inflamación , Masculino , Ratones , Receptores de Coronavirus/metabolismo , Transcriptoma , Replicación ViralRESUMEN
Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.
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Parásitos , Toxoplasma , Toxoplasmosis , Animales , Ratones , Plasmodium falciparumAsunto(s)
Manejo de la Enfermedad , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/sangre , Hipertensión Arterial Pulmonar/terapia , Circulación Pulmonar/fisiología , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Hipertensión Arterial Pulmonar/sangre , Hipertensión Arterial Pulmonar/fisiopatologíaRESUMEN
Leptospirosis is the leading cause of zoonotic morbidity and mortality globally, yet little is known about the immune mechanisms that may contribute to pathogenesis and severe disease. Although neutrophils are a key component of early immune responses to infection, they have been associated with tissue damage and inflammation in some febrile infections. To assess whether neutrophils contribute to the pathogenesis observed in severe leptospirosis, we quantitated levels of neutrophil activation markers in patients with varying disease severities. Hospitalized leptospirosis patients had significantly higher levels of toll-like receptors 2 and 4 (TLR2 and TLR4, respectively) on peripheral neutrophils than healthy controls, with the highest levels detected in patients with organ dysfunction. We observed no significant differences in other neutrophil baseline activation markers (CD62L and CD11b) or activation capacity (CD62L and CD11b levels following stimulation), regardless of disease severity. Our results provide preliminary evidence supporting the hypothesis that higher initial bacterial loads or inadequate or delayed neutrophil responses, rather than TLR-driven inflammation, may drive severe disease outcomes.
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Leptospirosis/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Adulto , Femenino , Humanos , Inflamación , Masculino , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Adulto JovenRESUMEN
We previously reported that the canonical innate immune receptor toll-like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic-specific, lung- and cell-targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4-/- mice. Lung Ec-silencing of TLR4 in wild-type mice induced emphysema, highlighting the specific and distinct role of Ec-expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16INK4a , a senescence-associated gene. Lung Ec-p16INK4a -silencing prevented TLR4-/- induced emphysema, revealing a new functional role for p16INK4a in lungs. TLR4 suppressed endogenous p16INK4a expression via HDAC2-mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence-related gene expression.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Células Endoteliales/metabolismo , Epigénesis Genética/genética , Pulmón/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor Toll-Like 4/deficienciaRESUMEN
Regulated cell death is a major mechanism to eliminate damaged, infected, or superfluous cells. Previously, apoptosis was thought to be the only regulated cell death mechanism; however, new modalities of caspase-independent regulated cell death have been identified, including necroptosis, pyroptosis, and autophagic cell death. As an understanding of the cellular mechanisms that mediate regulated cell death continues to grow, there is increasing evidence that these pathways are implicated in the pathogenesis of many pulmonary disorders. This review summarizes our understanding of regulated cell death as it pertains to the pathogenesis of chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and pulmonary arterial hypertension.