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Neoplasias Primarias Secundarias , Sarcoidosis , Seminoma , Neoplasias Testiculares , Masculino , Humanos , Seminoma/diagnóstico , Seminoma/patología , Seminoma/secundario , Sarcoidosis/diagnóstico , Tomografía Computarizada por Rayos X , Neoplasias Testiculares/diagnóstico , Neoplasias Testiculares/patología , Diagnóstico DiferencialRESUMEN
Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.
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Eosinofilia , Hipersensibilidad Inmediata , Hipersensibilidad , Células Madre Pluripotentes Inducidas , Niño , Animales , Humanos , Mutación con Ganancia de Función , Pez Cebra , Hipersensibilidad/genética , Inflamación/genética , Eosinofilia/genética , Janus Quinasa 1/genéticaRESUMEN
BACKGROUND: Flow cytometry is widely used for B-ALL minimal residual disease (MRD) analysis given its speed, availability, and sensitivity; however, distinguishing B-lymphoblasts from regenerative B-cells is not always straightforward. Radar plots, which project multiple markers onto a single plot, have been applied to other MRD analyses. Here we aimed to develop optimized radar plots for B-ALL MRD analysis. METHODS: We compiled Children's Oncology Group (COG) flow data from 20 MRD-positive and 9 MRD-negative B-ALL cases (enriched for hematogones) to create labeled training and test data sets with equal numbers of B-lymphoblasts, hematogones, and mature B-cells. We used an automated approach to create hundreds of radar plots and ranked them based on the ability of support vector machine (SVM) models to separate blasts from normal B-cells in the training data set. Top-performing radar plots were compared with PCA, t-SNE, and UMAP plots, evaluated with the test data set, and integrated into clinical workflows. RESULTS: SVM area under the ROC curve (AUC) for COG tube 1/2 radar plots improved from 0.949/0.921 to 0.989/0.968 after optimization. Performance was superior to PCA plots and comparable to UMAP, but with better generalizability to new data. When integrated into an MRD workflow, optimized radar plots distinguished B-lymphoblasts from other CD19-positive populations. MRD quantified by radar plots and serial gating were strongly correlated. DISCUSSION: Radar plots were successfully optimized to discriminate between diverse B-lymphoblast populations and non-malignant CD19-positive populations in B-ALL MRD analysis. Our novel radar plot optimization strategy could be adapted to other MRD panels and clinical scenarios.
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Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Enfermedad Aguda , Niño , Citometría de Flujo , Humanos , Aprendizaje Automático , Neoplasia Residual/diagnóstico , Neoplasia Residual/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , RadarAsunto(s)
Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Citometría de Flujo , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mieloide Aguda/genética , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: The coronavirus disease 2019 (COVID-19) pandemic raised concerns about the vulnerability of platelet supply and the uncertain impact of the resumption of elective surgery on utilization. We report the impact of COVID-19 on platelet supply and utilization across a large, integrated healthcare system in the Canadian province of British Columbia (BC). MATERIALS AND METHODS: Historical platelet use in BC by indication was compiled for fiscal year 2010/2011-2019/2020. Platelet collections, initial daily inventory and disposition data were assessed pre-COVID-19 (1 April 2018-15 March 2020) and for two COVID-19 time periods in BC: a shutdown phase with elective surgeries halted (16 March-17 May, 2020) and a renewal phase when elective surgeries resumed (18 May-27 September 2020); comparisons were made provincially and for individual health authorities. RESULTS: Historically, elective surgeries accounted for 10% of platelets transfused in BC. Initial daily supplier inventory increased from baseline during both COVID-19 periods (93/90 units vs. 75 units pre-COVID-19). During the shutdown phase, platelet utilization decreased 10.4% (41 units/week; p < 0.0001), and remained significantly decreased during the ensuing renewal period. Decreased platelet utilization was attributed to fewer transfusions during the shutdown phase followed by a decreased discard/expiry rate during the renewal phase compared to pre-COVID-19 (15.2% vs. 18.9% pre-COVID-19; p < 0.0001). Differences in COVID-19 platelet utilization patterns were noted between health authorities. CONCLUSION: Decreased platelet utilization was observed in BC compared to pre-COVID-19, likely due to a transient reduction in elective surgery as well as practice and policy changes triggered by pandemic concerns.
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COVID-19 , Plaquetas , Colombia Británica , Procedimientos Quirúrgicos Electivos , Humanos , SARS-CoV-2RESUMEN
Small interfering RNA (siRNA) therapy has significant potential for the treatment of myriad diseases, including cancer. While intravenous routes of delivery have been found to be effective for efficient targeting to the liver, achieving high accumulations selectively in other organs, including lung tissues, can be a challenge. We demonstrate the rational design and engineering of a layer-by-layer (LbL) nanoparticle-containing aerosol that is able to achieve efficient, multistage delivery of siRNA in vitro. For the purpose, LbL nanoparticles were, for the first time, encapsulated in composite porous micro scale particles using a supercritical CO2-assisted spray drying (SASD) apparatus using chitosan as an excipient. Such particles exhibited aerodynamic properties highly favorable for pulmonary administration, and effective silencing of mutant KRAS in lung cancer cells derived from tumors of a non-small cell lung cancer (NSCLC) autochthonous model. Furthermore, efficient alveolar accumulation following inhalation in healthy mice was also observed, corroborating in vitro aerodynamic results, and opening new perspectives for further studies of effective lung therapies These results show that multistage aerosols assembled by supercritical CO2-assisted spray drying can enable efficient RNA interference therapy of pulmonary diseases including lung cancer.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Nanopartículas , Administración por Inhalación , Aerosoles , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Excipientes , Pulmón , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
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Puntos de Control del Ciclo Celular/fisiología , Reparación del ADN/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Células HCT116 , Humanos , Immunoblotting , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Nanomedicina/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoAsunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Guanilato Ciclasa/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunodeficiencia Combinada Grave/genética , Niño , Humanos , Masculino , Mutación , Inmunodeficiencia Combinada Grave/diagnóstico , Secuenciación del Exoma/métodosRESUMEN
DNA damaging chemotherapy is a cornerstone of current front-line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo-resistant disease; therefore, adjuvant treatments that synergize with DNA-damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide-nucleic acid complex that facilitates tumor-specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex-mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high-grade serous OC to cytotoxic chemotherapy by blocking p38/MK2-dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.
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All-trans retinoic acid (ATRA), a derivative of vitamin A, is a common component in cosmetics and commercial acne creams as well as being a first-line chemotherapeutic agent. Today, formulations for the topical application of ATRA rely on creams and emulsions to incorporate the highly hydrophobic ATRA drug. These strategies, when applied to the skin, deliver ATRA as a single bolus, which is immediately taken up into the skin and contributes to many of the known adverse side effects of ATRA treatment, including skin irritation and hair loss. Herein we present a new concept in topical delivery of retinoids by covalently bonding the drug through a hydrolytically degradable ester linkage to a common hydrophilic polymer, polyvinyl alcohol (PVA), creating an amphiphilic nanomaterial that is water-soluble. This PVA bound ATRA can then act as a pro-drug and accumulate within the skin to allow for the sustained controlled delivery of active ATRA. This approach was demonstrated to release active ATRA out to 10days in vitro while significantly enhancing dermal accumulation of the ATRA in explant pig skin. In vivo we demonstrate that the pro-drug formulation reduces application site inflammation compared to free ATRA and retains the drug at the application site at measurable quantities for up to six days.
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Nanoestructuras/administración & dosificación , Alcohol Polivinílico/administración & dosificación , Profármacos/administración & dosificación , Tretinoina/administración & dosificación , Administración Cutánea , Animales , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos , Femenino , Ratones , Células 3T3 NIH , Nanoestructuras/química , Alcohol Polivinílico/química , Alcohol Polivinílico/farmacocinética , Profármacos/química , Profármacos/farmacocinética , Piel/metabolismo , Porcinos , Tretinoina/química , Tretinoina/farmacocinéticaRESUMEN
Layer-by-layer (LbL) self-assembly is a versatile technique from which multicomponent and stimuli-responsive nanoscale drug carriers can be constructed. Despite the benefits of LbL assembly, the conventional synthetic approach for fabricating LbL nanoparticles requires numerous purification steps that limit scale, yield, efficiency, and potential for clinical translation. In this report, we describe a generalizable method for increasing throughput with LbL assembly by using highly scalable, closed-loop diafiltration to manage intermediate purification steps. This method facilitates highly controlled fabrication of diverse nanoscale LbL formulations smaller than 150 nm composed from solid-polymer, mesoporous silica, and liposomal vesicles. The technique allows for the deposition of a broad range of polyelectrolytes that included native polysaccharides, linear polypeptides, and synthetic polymers. We also explore the cytotoxicity, shelf life and long-term storage of LbL nanoparticles produced using this approach. We find that LbL coated systems can be reliably and rapidly produced: specifically, LbL-modified liposomes could be lyophilized, stored at room temperature, and reconstituted without compromising drug encapsulation or particle stability, thereby facilitating large scale applications. Overall, this report describes an accessible approach that significantly improves the throughput of nanoscale LbL drug-carriers that show low toxicity and are amenable to clinically relevant storage conditions.
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RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.
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ADN Circular/metabolismo , ARN Interferente Pequeño/biosíntesis , Ribonucleasa III/genética , Línea Celular Tumoral , Silenciador del Gen , Ingeniería Genética/métodos , Humanos , Conformación de Ácido Nucleico , Poliaminas/química , Polielectrolitos , ARN Interferente Pequeño/química , Transducción GenéticaRESUMEN
Packaging multiple small interfering RNA (siRNA) molecules into nanostructures at precisely defined ratios is a powerful delivery strategy for effective RNA interference (RNAi) therapy. We present a novel RNA nanotechnology based approach to produce multiple components of polymerized siRNA molecules that are simultaneously self-assembled and densely packaged into composite sponge-like porous microstructures (Multi-RNAi-MSs) by rolling circle transcription. The Multi-RNAi-MSs were designed to contain a combination of multiple polymeric siRNA molecules with precisely controlled stoichiometry within a singular microstructure by manipulating the types and ratios of the circular DNA templates. The Multi-RNAi-MSs were converted into nanosized complexes by polyelectrolyte condensation to manipulate their physicochemical properties (size, shape, and surface charge) for favorable delivery, while maintaining the multifunctional properties of the siRNAs for combined therapeutic effects. These Multi-RNAi-MS systems have great potential in RNAi-mediated biomedical applications, for example, for the treatment of cancer, genetic disorders, and viral infections.
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Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Microscopía Electrónica de RastreoRESUMEN
Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.
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Antineoplásicos/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Antineoplásicos/inmunología , Antineoplásicos/metabolismo , Secuencia de Bases , Caspasa 3/genética , Caspasa 3/inmunología , Caspasa 7/genética , Caspasa 7/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Humanos , Inmunidad Innata , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Luciferasas/inmunología , Datos de Secuencia Molecular , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Conformación de Ácido Nucleico , Poli I-C/genética , Poli I-C/inmunología , Poli I-C/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Transcripción GenéticaRESUMEN
PURPOSE: Cross-talk and feedback between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR cell signaling pathways is critical for tumor initiation, maintenance, and adaptive resistance to targeted therapy in a variety of solid tumors. Combined blockade of these pathways-horizontal blockade-is a promising therapeutic strategy; however, compounded dose-limiting toxicity of free small molecule inhibitor combinations is a significant barrier to its clinical application. EXPERIMENTAL DESIGN: AZD6244 (selumetinib), an allosteric inhibitor of Mek1/2, and PX-866, a covalent inhibitor of PI3K, were co-encapsulated in a tumor-targeting nanoscale drug formulation-layer-by-layer (LbL) nanoparticles. Structure, size, and surface charge of the nanoscale formulations were characterized, in addition to in vitro cell entry, synergistic cell killing, and combined signal blockade. In vivo tumor targeting and therapy was investigated in breast tumor xenograft-bearing NCR nude mice by live animal fluorescence/bioluminescence imaging, Western blotting, serum cytokine analysis, and immunohistochemistry. RESULTS: Combined MAPK and PI3K axis blockade from the nanoscale formulations (160 ± 20 nm, -40 ± 1 mV) was synergistically toxic toward triple-negative breast (MDA-MB-231) and RAS-mutant lung tumor cells (KP7B) in vitro, effects that were further enhanced upon encapsulation. In vivo, systemically administered LbL nanoparticles preferentially targeted subcutaneous MDA-MB-231 tumor xenografts, simultaneously blocked tumor-specific phosphorylation of the terminal kinases Erk and Akt, and elicited significant disease stabilization in the absence of dose-limiting hepatotoxic effects observed from the free drug combination. Mice receiving untargeted, but dual drug-loaded nanoparticles exhibited progressive disease. CONCLUSIONS: Tumor-targeting nanoscale drug formulations could provide a more safe and effective means to synergistically block MAPK and PI3K in the clinic.
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Antineoplásicos/administración & dosificación , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Nanopartículas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Nanopartículas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.
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Antineoplásicos/administración & dosificación , ADN sin Sentido/química , Portadores de Fármacos , Microscopía Electrónica de TransmisiónRESUMEN
Active targeting of nanoscale drug carriers can improve tumor-specific delivery; however, cellular heterogeneity both within and among tumor sites is a fundamental barrier to their success. Here, we describe a tumor microenvironment-responsive layer-by-layer (LbL) polymer drug carrier that actively targets tumors based on two independent mechanisms: pH-dependent cellular uptake at hypoxic tumor pH and hyaluronan-directed targeting of cell-surface CD44 receptor, a well-characterized biomarker for breast and ovarian cancer stem cells. Hypoxic pH-induced structural reorganization of hyaluronan-LbL nanoparticles was a direct result of the nature of the LbL electrostatic complex, and led to targeted cellular delivery in vitro and in vivo, with effective tumor penetration and uptake. The nanoscale drug carriers selectively bound CD44 and diminished cancer cell migration in vitro, while co-localizing with the CD44 receptor in vivo. Multimodal targeting of LbL nanoparticles is a powerful strategy for tumor-specific cancer diagnostics and therapy that can be accomplished using a single bilayer of polyamine and hyaluronan that, when assembled, produce a dynamic and responsive cell-particle interface.