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Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (â¼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying â¼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.
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Virus Hendra , Virus Nipah , Células Madre Pluripotentes , Arterias , Células Endoteliales , Virus Hendra/genética , Humanos , TropismoRESUMEN
T cell receptor (TCR) recognition of a peptide-major histocompatibility complex (pMHC) is crucial for adaptive immune response. The identification of therapeutically relevant TCR-pMHC protein pairs is a bottleneck in the implementation of TCR-based immunotherapies. The ability to computationally design TCRs to target a specific pMHC requires automated integration of next-generation sequencing, protein-protein structure prediction, molecular dynamics, and TCR ranking. A pipeline to evaluate patient-specific, sequence-based TCRs to a target pMHC is presented. Using the three most frequently expressed TCRs from 16 colorectal cancer patients, the protein-protein structure of the TCRs to the target CEA peptide-MHC is predicted using Modeller and ColabFold. TCR-pMHC structures are compared using automated equilibration and successive analysis. ColabFold generated configurations require an ≈2.5× reduction in equilibration time of TCR-pMHC structures compared to Modeller. The structural differences between Modeller and ColabFold are demonstrated by root mean square deviation (≈0.20 nm) between clusters of equilibrated configurations, which impact the number of hydrogen bonds and Lennard-Jones contacts between the TCR and pMHC. TCR ranking criteria that may prioritize TCRs for evaluation of in vitro immunogenicity are identified, and this ranking is validated by comparing to state-of-the-art machine learning-based methods trained to predict the probability of TCR-pMHC binding.
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The coordinated (dis)engagement of the membrane-bound T cell receptor (TCR)-CD3-CD4 complex from the peptide-major histocompatibility complex (pMHC) is fundamental to TCR signal transduction and T cell effector function. As such, an atomic-scale understanding would not only enhance our basic understanding of the adaptive immune response but would also accelerate the rational design of TCRs for immunotherapy. In this study, we explore the impact of the CD4 coreceptor on the TCR-pMHC (dis)engagement by constructing a molecular-level biomimetic model of the CD3-TCR-pMHC and CD4-CD3-TCR-pMHC complexes within a lipid bilayer. After allowing the system complexes to equilibrate (engage), we use steered molecular dynamics to dissociate (disengage) the pMHC. We find that 1) the CD4 confines the pMHC closer to the T cell by 1.8 nm at equilibrium; 2) CD4 confinement shifts the TCR along the MHC binding groove engaging a different set of amino acids and enhancing the TCR-pMHC bond lifetime; 3) the CD4 translocates under load increasing the interaction strength between the CD4-pMHC, CD4-TCR, and CD4-CD3; and 4) upon dissociation, the CD3-TCR complex undergoes structural oscillation and increased energetic fluctuation between the CD3-TCR and CD3-lipids. These atomic-level simulations provide mechanistic insight on how the CD4 coreceptor impacts TCR-pMHC (dis)engagement. More specifically, our results provide further support (enhanced bond lifetime) for a force-dependent kinetic proofreading model and identify an alternate set of amino acids in the TCR that dominate the TCR-pMHC interaction and could thus impact the design of TCRs for immunotherapy.
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Biomimética , Receptores de Antígenos de Linfocitos T , Complejo CD3/química , Complejo CD3/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Complejo Mayor de Histocompatibilidad , Péptidos/química , Simulación de Dinámica Molecular , Unión Proteica , Aminoácidos/metabolismoRESUMEN
Recreating human organ-level function in vitro is a rapidly evolving field that integrates tissue engineering, stem cell biology, and microfluidic technology to produce 3D organoids. A critical component of all organs is the vasculature. Herein, we discuss general strategies to create vascularized organoids, including common source materials, and survey previous work using vascularized organoids to recreate specific organ functions and simulate tumor progression. Vascularization is not only an essential component of individual organ function but also responsible for coupling the fate of all organs and their functions. While some success in coupling two or more organs together on a single platform has been demonstrated, we argue that the future of vascularized organoid technology lies in creating organoid systems complete with tissue-specific microvasculature and in coupling multiple organs through a dynamic vascular network to create systems that can respond to changing physiological conditions.
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Dispositivos Laboratorio en un Chip , Organoides , Humanos , Microfluídica , Células Madre , Ingeniería de TejidosRESUMEN
Convective transport can significantly distort spatial concentration gradients. Interstitial flow is ubiquitous throughout living tissue, but our understanding of how interstitial flow affects concentration gradients in biological processes is limited. Interstitial flow is of particular interest for angiogenesis because pathological and physiological angiogenesis is associated with altered interstitial flow, and both interstitial flow and morphogen gradients (e.g., vascular endothelial growth factor, VEGF) can potentially stimulate and guide new blood vessel growth. We designed an in vitro microfluidic platform to simulate 3D angiogenesis in a tissue microenvironment that precisely controls interstitial flow and spatial morphogen gradients. The microvascular tissue was developed from endothelial colony forming cell-derived endothelial cells extracted from cord blood and stromal fibroblasts in a fibrin extracellular matrix. Pressure in the microfluidic lines was manipulated to control the interstitial flow. A mathematical model of mass and momentum transport, and experimental studies with fluorescently labeled dextran were performed to validate the platform. Our data demonstrate that at physiological interstitial flow (0.1-10 µm/s), morphogen gradients were eliminated within hours, and angiogenesis demonstrated a striking bias in the opposite direction of interstitial flow. The interstitial flow-directed angiogenesis was dependent on the presence of VEGF, and the effect was mediated by αvß3 integrin. We conclude that under physiological conditions, growth factors such as VEGF and fluid forces work together to initiate and spatially guide angiogenesis.
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Líquido Extracelular/fisiología , Neovascularización Fisiológica , Difusión , Humanos , Integrina alfaVbeta3/metabolismo , Microfluídica , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.
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Factores de Transcripción/metabolismo , Células Cultivadas , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunohistoquímica , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metilcelulosa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción de la Familia SnailRESUMEN
Human pluripotent stem cell-derived cardiomyocytes (hPS-CM) may offer a number of advantages over previous cardiac models, however, questions of their immaturity complicate their adoption as a new in vitro model. hPS-CM differ from adult cardiomyocytes with respect to structure, proliferation, metabolism and electrophysiology, better approximating fetal cardiomyocytes. Time in culture appears to significantly impact phenotype, leading to what can be referred to as early and late hPS-CM. This work surveys the phenotype of hPS-CM, including structure, bioenergetics, sensitivity to damage, gene expression, and electrophysiology, including action potential, ion channels, and intracellular calcium stores, while contrasting fetal and adult CM with hPS-CM at early and late time points after onset of differentiation.
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Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Humanos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
BACKGROUND: We previously showed that impulse oscillometry (IOS) indices of peripheral airway function are associated with asthma control in children. However, little data exist on whether dysfunction in the peripheral airways can predict loss of asthma control. OBJECTIVE: We sought to determine the utility of peripheral airway impairment, as measured by IOS, in predicting loss of asthma control in children. METHODS: Fifty-four children (age, 7-17 years) with controlled asthma were enrolled in the study. Spirometric and IOS indices of airway function were obtained at baseline and at a follow-up visit 8 to 12 weeks later. Physicians who were blinded to the IOS measurements assessed asthma control (National Asthma Education and Prevention Program guidelines) on both visits and prescribed no medication change between visits. RESULTS: Thirty-eight (70%) patients maintained asthma control between 2 visits (group C-C), and 16 patients had asthma that became uncontrolled on the follow-up visit (group C-UC). There was no difference in baseline spirometric results between the C-C and C-UC groups, except for FEV1/forced vital capacity ratio (86% vs 82%, respectively; P < .01). Baseline IOS results, including resistance of the respiratory system at 5 Hz (R5; 6.4 vs 4.3 cm H2O · L(-1) · s), frequency dependence of resistance (difference of R5 and resistance of the respiratory system at 20 Hz [R5-20]; 2.0 vs 0.7 cm H2O · L(-1) · s), and reactance area (13.1 vs 4.1 cm H2O · L(-1)), of group C-UC were significantly higher than those of group C-C (P < .01). Receiver operating characteristic analysis showed baseline R5-20 and reactance area effectively predicted asthma control status at the follow-up visit (area under the curve, 0.91 and 0.90). CONCLUSION: Children with controlled asthma who have increased peripheral airway IOS indices are at risk of losing asthma control.
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Resistencia de las Vías Respiratorias , Asma/fisiopatología , Adolescente , Asma/diagnóstico , Niño , Femenino , Humanos , Pulmón/fisiopatología , Masculino , Oscilometría , EspirometríaRESUMEN
The hemorrhagic fever viruses (HFVs) cause severe or fatal infections in humans. Named after their common symptom hemorrhage, these viruses induce significant vascular dysfunction by affecting endothelial cells, altering immunity, and disrupting the clotting system. Despite advances in treatments, such as cytokine blocking therapies, disease modifying treatment for this class of pathogen remains elusive. Improved understanding of the pathogenesis of these infections could provide new avenues to treatment. While animal models and traditional 2D cell cultures have contributed insight into the mechanisms by which these pathogens affect the vasculature, these models fall short in replicatingin vivohuman vascular dynamics. The emergence of microphysiological systems (MPSs) offers promising avenues for modeling these complex interactions. These MPS or 'organ-on-chip' models present opportunities to better mimic human vascular responses and thus aid in treatment development. In this review, we explore the impact of HFV on the vasculature by causing endothelial dysfunction, blood clotting irregularities, and immune dysregulation. We highlight how existing MPS have elucidated features of HFV pathogenesis as well as discuss existing knowledge gaps and the challenges in modeling these interactions using MPS. Understanding the intricate mechanisms of vascular dysfunction caused by HFV is crucial in developing therapies not only for these infections, but also for other vasculotropic conditions like sepsis.
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Fiebres Hemorrágicas Virales , Humanos , Fiebres Hemorrágicas Virales/virología , Animales , Células Endoteliales/patología , Endotelio Vascular , Modelos BiológicosRESUMEN
Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byßcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletßcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingßcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.
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Diabetes Mellitus , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Humanos , Insulina/metabolismo , Diabetes Mellitus/metabolismo , Glucosa/metabolismoRESUMEN
Epithelial injury and airway hyperresponsiveness are prominent features of asthma. We have previously demonstrated that laser ablation of single epithelial cells immediately induces global airway constriction through Ca(2+)-dependent smooth muscle shortening. The response is mediated by soluble mediators released from wounded single epithelial cells; however, the soluble mediators and signaling mechanisms have not been identified. In this study, we investigated the nature of the epithelial-derived soluble mediators and the associated signaling pathways that lead to the L-type voltage-dependent Ca(2+) channel (VGCC)-mediated Ca(2+) influx. We found that inhibition of adenosine A1 receptors (or removal of adenosine with adenosine deaminase), cyclooxygenase (COX)-2 or prostaglandin E receptor 3 (EP3) receptors, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) all significantly blocked Ca(2+) oscillations in smooth muscle cells and airway contraction induced by local epithelial injury. Using selective agonists to activate the receptors in the presence and absence of selective receptor antagonists, we found that adenosine activated the signaling pathway A1RâEGFR/PDGFRâCOX-2âEP3âVGCCsâcalcium-induced calcium release, leading to intracellular Ca(2+) oscillations in airway smooth muscle cells and airway constriction.
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Contracción Muscular/fisiología , Receptor de Adenosina A1/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Mucosa Respiratoria/metabolismo , Sistema Respiratorio/fisiopatología , Heridas y Lesiones/fisiopatología , Antagonistas del Receptor de Adenosina A1/farmacología , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/antagonistas & inhibidores , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/lesiones , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Heridas y Lesiones/metabolismoRESUMEN
BACKGROUND: Previous reports suggest that the peripheral airways are associated with asthma control. Patient history, although subjective, is used largely to assess asthma control in children because spirometric results are many times normal values. Impulse oscillometry (IOS) is an objective and noninvasive measurement of lung function that has the potential to examine independently both small- and large-airway obstruction. OBJECTIVE: We sought to determine the utility of IOS in assessing asthma control in children. METHODS: Asthmatic and healthy children (6-17 years) were enrolled in the study. Spirometric and IOS (resistance of the respiratory system at 5 Hz [R5] and 20 Hz [R20], reactance of the respiratory system at 5 Hz [X5], resonant frequency of reactance [Fres], and area under the reactance curve between 5 Hz and Fres [reactance area {AX}]) values were collected in triplicate before and after a bronchodilator was administered. The physicians were blinded to the IOS measurements and assessed asthma control using American Thoracic Society guidelines. RESULTS: Small-airway IOS measurements, including the difference of R5 and R20 [R5-20], X5, Fres, and AX, of children with uncontrolled asthma (n = 44) were significantly different from those of children with controlled asthma (n = 57) and healthy children (n = 14), especially before the administration of a bronchodilator. However, there was no difference in large-airway IOS values (R20). No differences were found between children with controlled asthma and healthy children in any of the end points. Receiver operating characteristic analysis showed cut points for baseline R5-20 (1.5 cm H(2)O · L(-1) · s) and AX (9.5 cm H(2)O · L(-1)) that effectively discriminated controlled versus uncontrolled asthma (area under the curve, 0.86 and 0.84) and correctly classified more than 80% of the population. CONCLUSION: Uncontrolled asthma is associated with small-airways dysfunction, and IOS might be a reliable and noninvasive method to assess asthma control in children.
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Asma/diagnóstico , Asma/patología , Pulmón/metabolismo , Oscilometría , Pruebas de Función Respiratoria , Adolescente , Asma/fisiopatología , Asma/prevención & control , Niño , Erradicación de la Enfermedad , Estudios de Factibilidad , Femenino , Humanos , Pulmón/patología , Masculino , Pruebas de Función Respiratoria/métodos , EspirometríaRESUMEN
BACKGROUND: The importance of monitoring exhaled nitric oxide (NO) in asthma remains controversial. OBJECTIVE: To measure exhaled NO, postnebulized albuterol/ipratropium spirometry, and Asthma Control Test (ACT) during asthma exacerbation requiring 8- to 10-day tapering oral corticosteroid in nonsmoking patients with moderate-to-severe asthma on moderate-dose inhaled corticosteroid and long-acting ß(2)-agonist but not maintenance oral corticosteroid. METHODS: After measuring the fraction of exhaled NO (Feno [ppb]) at 50, 100, 150, and 200 mL/s, the total Feno at 50 mL/s (ppb), large central airway NO flux (J'(awNO) [nL/s]), and peripheral small airway/alveolar NO concentration (C(ANO) [ppb]) were calculated and corrected for NO axial back-diffusion. Outpatient exacerbation required the patient with asthma to be afebrile with normal chest x-ray and white blood cell count. RESULTS: Group 1 included 17 patients (6 men) with asthma, age 52 ± 12 years, studied at baseline, during 18 exacerbations with abnormal Feno at 50 mL/s, J'(awNO), and/or C(ANO), and post 8- to 10-day tapering 40 mg prednisone (recovery). Baseline: IgE, 332 ± 243 Kµ; total blood eosinophils, 304 ± 266 cells/µL; body mass index, 28 ± 6; ACT, 16 to 19; and FEV(1), 2.5 ± 0.7 L (86% ± 20% predicted); exacerbation: FEV(1), 1.7 ± 0.4 L (60% ± 17%) (P < .001); recovery: FEV(1), 2.5 ± 0.7 L (85% ± 13%) (P < .001). Group 2 included 11 (7 men) similarly treated patients with asthma, age 49 ± 14 years, studied at baseline, during 15 exacerbations with normal Feno at 50 mL/s, J'(awNO), and C(ANO). Baseline: IgE, 307 ± 133 Kµ; total blood eosinophils, 296 ± 149 cells/µL; body mass index, 28 ± 6; ACT, 16 to 19; and FEV(1), 2.7 ± 0.9 L (71% ± 12% predicted); exacerbation: FEV(1), 1.7 ± 0.6 L (54% ± 19%) (P< .006); recovery: FEV(1), 2.7 ± 0.9 L (70% ± 14%) (P= .002). On comparing group 1 versus group 2, there was no significant difference for baseline IgE, eosinophils, body mass index, and ACT and similar significant (≤.006) decrease from baseline in FEV(1) (L) during exacerbation and similar increase (≤.006) at recovery. CONCLUSIONS: Increased versus normal exhaled NO during outpatient exacerbation in patients with moderate-to-severe asthma on inhaled corticosteroid and long-acting ß(2)-agonist but not maintenance oral corticosteroid does not preclude a robust clinical and spirometric response to tapering oral prednisone.
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Corticoesteroides/uso terapéutico , Asma/diagnóstico , Asma/tratamiento farmacológico , Óxido Nítrico/análisis , Administración Oral , Corticoesteroides/administración & dosificación , Adulto , Anciano , Progresión de la Enfermedad , Espiración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Pruebas de Función Respiratoria , Espirometría , Factores de Tiempo , Resultado del TratamientoRESUMEN
Assessing B cell affinity to pathogen-specific antigens prior to or following exposure could facilitate the assessment of immune status. Current standard tools to assess antigen-specific B cell responses focus on equilibrium binding of the secreted antibody in serum. These methods are costly, time-consuming, and assess antibody affinity under zero-force. Recent findings indicate that force may influence BCR-antigen binding interactions and thus immune status. Here, we designed a simple laminar flow microfluidic chamber in which the antigen (hemagglutinin of influenza A) is bound to the chamber surface to assess antigen-specific BCR binding affinity of five hemagglutinin-specific hybridomas under 65- to 650-pN force range. Our results demonstrate that both increasing shear force and bound lifetime can be used to enrich antigen-specific high affinity B cells. The affinity of the membrane-bound BCR in the flow chamber correlates well with the affinity of the matched antibodies measured in solution. These findings demonstrate that a microfluidic strategy can rapidly assess BCR-antigen binding properties and identify antigen-specific high affinity B cells. This strategy has the potential to both assess functional immune status from peripheral B cells and be a cost-effective way of identifying individual B cells as antibody sources for a range of clinical applications.
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A feature of severe COVID-19 is the onset of an acute and intense systemic inflammatory response referred to as the "cytokine storm". The cytokine storm is characterized by high serum levels of inflammatory cytokines and the subsequent transport of inflammatory cells to damaging levels in vital organs (e.g., myocarditis). Immune trafficking and its effect on underlying tissues (e.g., myocardium) are challenging to observe at a high spatial and temporal resolution in mouse models. In this study, we created a vascularized organ-on-a-chip system to mimic cytokine storm-like conditions and tested the effectiveness of a novel multivalent selectin-targeting carbohydrate conjugate (composed of DS - dermatan sulfate and IkL - a selectin-binding peptide, termed DS-IkL) in blocking infiltration of polymorphonuclear leukocytes (PMN). Our data shows that cytokine storm-like conditions induce endothelial cells to produce additional inflammatory cytokines and facilitate infiltration of PMNs into tissue. Treatment of tissues with DS-IkL (60 µM) reduced PMN accumulation in the tissue by >50%. We then created cytokine storm-like conditions in a vascularized cardiac tissue-chip and found that PMN infiltration increases the spontaneous beating rate of the cardiac tissue, and this effect is eliminated by treatment with DS-IkL (60 µM). In summary, we demonstrate the utility of an organ-on-a-chip platform to mimic COVID-19 related cytokine storm and that blocking leukocyte infiltration with DS-IkL could be a viable strategy to mitigate associated cardiac complications.
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COVID-19 , Neutrófilos , Ratones , Animales , Cardiotoxicidad , Células Endoteliales , Sistemas Microfisiológicos , CitocinasRESUMEN
Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (â¼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.
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Vesículas Extracelulares , Laminina , Humanos , Laminina/metabolismo , Convección , Integrina alfa6beta1/metabolismo , Vesículas Extracelulares/metabolismo , Integrina alfa3beta1/metabolismo , Matriz Extracelular/metabolismoRESUMEN
ALK1 (ACVRL1) is a member of the TGFß receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for hereditary hemorrhagic telangiectasia type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations. Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1-restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.
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Receptores de Activinas Tipo I/genética , Endotelio Vascular/metabolismo , Efrina-B2/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Telangiectasia Hemorrágica Hereditaria/metabolismo , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animales , Secuencia de Bases , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Cartilla de ADN , Endotelio Vascular/citología , Efrina-B2/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Transducción de Señal , Telangiectasia Hemorrágica Hereditaria/genéticaRESUMEN
BACKGROUND: The Childhood Asthma Control Test (C-ACT) and the Asthma Control Test (ACT) are validated measures of asthma control in which a score of 19 is defined as uncontrolled according to published reports. However, different cut points may exist in different ethnic populations. OBJECTIVE: To determine the cut point for uncontrolled asthma in a Mexican descent population from Orange Country, California, compared with an age- and asthma severity-matched non-Hispanic cohort. METHODS: The C-ACT (in children 6-11 years old) and ACT (in children 12-17 years old) scores were collected from 151 children of Mexican descent and 48 non-Hispanic controls with mild-to-moderate asthma who lived in Orange County. Physicians were masked to C-ACT and ACT scores while assessing control based on National Asthma Education and Prevention program guidelines. The receiver operating characteristic method was used to examine the screening accuracy of the tests to detect uncontrolled asthma. The optimal cut points were selected by maximizing the total sensitivity and specificity. RESULTS: Cronbach α values for the C-ACT (0.76) and the ACT (0.80) confirmed that both tests were reliable in our study population. The C-ACT and ACT scores were statistically higher in children of Mexican descent than non-Hispanic children (P = .008). A cut point of 22 was optimal to detect uncontrolled asthma in children of Mexican descent 6 to 11 years old (group 1: sensitivity, 0.74; specificity, 0.86; area under the curve [AUC], 0.83) and children 12 to 17 years old (group 3: sensitivity, 0.78; specificity, 0.68; AUC, 0.79). For non-Hispanic controls, a cut point of 20 were optimal to detect uncontrolled asthma in children 6 to 11 years old (group 2: sensitivity, 0.70; specificity, 0.91; AUC, 0.86) and children 12 to 17 years old (group 4: sensitivity, 0.83; specificity, 0.87; AUC, 0.91). CONCLUSION: In this cross-ethnic validation study, children of Mexican descent in Orange County seem to underreport asthma symptoms compared with a non-Hispanic population and may require higher C-ACT and ACT cut points to detect uncontrolled asthma.
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Asma/diagnóstico , Asma/epidemiología , Americanos Mexicanos , Índice de Severidad de la Enfermedad , Adolescente , California/epidemiología , Niño , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Americanos Mexicanos/estadística & datos numéricos , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
An atomic-scale mechanism of T Cell Receptor (TCR) mechanosensing of peptides in the binding groove of the peptide-major histocompatibility complex (pMHC) may inform the design of novel TCRs for immunotherapies. Using steered molecular dynamics simulations, our study demonstrates that mutations to peptides in the binding groove of the pMHC - which are known to discretely alter the T cell response to an antigen - alter the MHC conformation at equilibrium. This subsequently impacts the overall strength (duration and length) of the TCR-pMHC bond under constant load. Moreover, physiochemical features of the TCR-pMHC dynamic bond strength, such as hydrogen bonds and Lennard-Jones contacts, correlate with the immunogenic response elicited by the specific peptide in the MHC groove. Thus, formation of transient TCR-pMHC bonds is characteristic of immunogenic peptides, and steered molecular dynamics simulations can be used in the overall design strategy of TCRs for immunotherapies.
RESUMEN
Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and customized video-microscopy tracking tools. The recent development of Trackmate has increased researcher accessibility to tracking particles in video-microscopy experiments; however, there is a lack of tools that analyze this tracking information. We report a software tool, compatible with Trackmate, that extracts Receptor Ligand Non-Equilibrium Kinetic (RLNEK) parameters from video-microscopy data. This software should be of particular interest to the community of researchers and scientists interrogating the target-specific binding and release of immune cells.