RESUMEN
Our study explores the role of cancer-derived extracellular exosomes (EXs), particularly focusing on collagen alpha-3 (VI; COL6A3), in facilitating tumor dissemination and metastasis in epithelial ovarian cancer (EOC). We found that COL6A3 is expressed in aggressive ES2 derivatives, SKOV3 overexpressing COL6A3 (SKOV3/COL6A3), and mesenchymal-type ovarian carcinoma stromal progenitor cells (MSC-OCSPCs), as well as their EXs, but not in less aggressive SKOV3 cells or ES2 cells with COL6A3 knockdown (ES2/shCOL6A3). High COL6A3 expression correlates with worse overall survival among EOC patients, as evidenced by TCGA and GEO data analysis. In vitro experiments showed that EXs from MSC-OCSPCs or SKOV3/COL6A3 cells significantly enhance invasion ability in ES2 or SKOV3/COL6A3 cells, respectively (both, p <0.001). In contrast, ES2 cells with ES2/shCOL6A3 EXs exhibited reduced invasion ability (p < 0.001). In vivo, the average disseminated tumor numbers in the peritoneal cavity were significantly greater in mice receiving intraperitoneally injected SKOV3/COL6A3 cells than in SKOV3 cells (p < 0.001). Furthermore, mice intravenously (IV) injected with SKOV3/COL6A3 cells and SKOV3/COL6A3-EXs showed increased lung colonization compared to mice injected with SKOV3 cells and PBS (p = 0.007) or SKOV3/COL6A3 cells and PBS (p = 0.039). Knockdown of COL6A3 or treatment with EX inhibitor GW4869 or rapamycin-abolished COL6A3-EXs may suppress the aggressiveness of EOC.
Asunto(s)
Carcinoma Epitelial de Ovario , Colágeno Tipo VI , Exosomas , Neoplasias Ováricas , Exosomas/metabolismo , Exosomas/genética , Femenino , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Humanos , Colágeno Tipo VI/metabolismo , Colágeno Tipo VI/genética , Animales , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratones , Línea Celular Tumoral , Metástasis de la Neoplasia , Invasividad Neoplásica , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Movimiento CelularRESUMEN
Tuberculosis (TB) remains a major global public health problem, and improved treatments are needed to shorten duration of therapy, decrease disease burden, improve compliance, and combat emergence of drug resistance. Ideally, the most effective regimen would be identified by a systematic and comprehensive combinatorial search of large numbers of TB drugs. However, optimization of regimens by standard methods is challenging, especially as the number of drugs increases, because of the extremely large number of drug-dose combinations requiring testing. Herein, we used an optimization platform, feedback system control (FSC) methodology, to identify improved drug-dose combinations for TB treatment using a fluorescence-based human macrophage cell culture model of TB, in which macrophages are infected with isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible green fluorescent protein (GFP)-expressing Mycobacterium tuberculosis (Mtb). On the basis of only a single screening test and three iterations, we identified highly efficacious three- and four-drug combinations. To verify the efficacy of these combinations, we further evaluated them using a methodologically independent assay for intramacrophage killing of Mtb; the optimized combinations showed greater efficacy than the current standard TB drug regimen. Surprisingly, all top three- and four-drug optimized regimens included the third-line drug clofazimine, and none included the first-line drugs isoniazid and rifampin, which had insignificant or antagonistic impacts on efficacy. Because top regimens also did not include a fluoroquinolone or aminoglycoside, they are potentially of use for treating many cases of multidrug- and extensively drug-resistant TB. Our study shows the power of an FSC platform to identify promising previously unidentified drug-dose combinations for treatment of TB.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/administración & dosificación , Línea Celular , Células Cultivadas , Combinación de Medicamentos , Interacciones Farmacológicas , Retroalimentación , Proteínas Fluorescentes Verdes/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Isopropil Tiogalactósido/genética , Macrófagos/microbiología , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológicoRESUMEN
Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation (p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis (p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly (p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Algoritmos , Animales , Apoptosis , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Pollos , Membrana Corioalantoides/metabolismo , Cimenos , Ensayos de Selección de Medicamentos Antitumorales , Células Endoteliales/citología , Retroalimentación , Femenino , Humanos , Imidazoles/administración & dosificación , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Compuestos Organometálicos/administración & dosificación , Neoplasias Ováricas/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Quinolinas/administración & dosificación , Procesos EstocásticosRESUMEN
BACKGROUND: This study aimed to investigate whether the DNA methylation of human ovarian carcinoma stromal progenitor cells (OCSPCs) could promote the tumorigenesis of ovarian carcinoma. METHODS: OCSPCs were first isolated from fresh tumor tissues and ascites of ovarian cancer patients. In vivo and in vitro experiments on the effect of the OCSPCs on tumorigenesis and the effects of DNA demethylation on the OCSPCs were then performed. RESULTS: The OCSPCs possessed self-renewal and multipotent differentiation capacity with elevated expressions of OCT4, NANOG, BMP2, BMP4, Rex-1, AC133 and TGF-ß. The OCSPCs, when combined with tumor cells in vivo could promote tumor growth. The methylation profiles of tumor suppressor genes (TSGs) were significantly higher in the OCSPCs than in ovarian cancer cells (p < 0.001). 5-aza-2-dC could alter the methylation levels of TSGs in OCSPCs and also inhibit the tumor promoting capabilities of the OCSPCs by decreasing the proliferation of tumors cells. The expression levels of TSGs were re-expressed by 5-aza-2-dC to inhibit the self-renewal and growth of OCSPCs. CONCLUSIONS: OCSPCs with decreased TSG expressions in the ovarian tumor microenvironment were able to promote tumorigenesis which could be reversed by DNA demethylation. DNA demethylation reversing the expression of TSGs in OCSPCs may represent a potential therapeutic target for ovarian cancer.
Asunto(s)
Carcinogénesis , Metilación de ADN , Neoplasias Ováricas/genética , Células del Estroma/patología , Animales , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Linaje de la Célula , Transición Epitelial-Mesenquimal , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patologíaRESUMEN
We compared multiplex E6 messenger ribonucleic acid (mRNA) tests using real-time quantitative reverse transcriptase polymerase chain reactions (PCR) with human papillomavirus (HPV) DNA subtypes using a MY11/GP6+ PCR-based reverse-blot assay to identify cervical intraepithelial neoplasias of grade 2 or worse (CIN2+). In total, 684 women were studied, of whom 377 (55%) were diagnosed with CIN2+ histologically. The specificity of HPV mRNA to predict histological CIN2+ was higher than that of HPV DNA (81.3% vs. 44.2%). The odds ratios (ORs) to predict histological CIN2+ in women with positive for type 16, 18, 31, and 45 E6 mRNA or by HPV DNA detection were 7.1 (95% confidence interval [CI] 3.9-13.1) and 2.5 (95%CI 1.9-3.5), respectively, compared to those with negative for E6 mRNA or HPV DNA. The OR to predict histological CIN2+ in women with a cytological grade Asunto(s)
ADN Viral/análisis
, Tamizaje Masivo/métodos
, Técnicas de Diagnóstico Molecular/métodos
, Proteínas Oncogénicas Virales/análisis
, Infecciones por Papillomavirus/complicaciones
, ARN Mensajero/análisis
, Displasia del Cuello del Útero/diagnóstico
, Adulto
, Anciano
, Anciano de 80 o más Años
, Estudios Transversales
, ADN Viral/genética
, Femenino
, Humanos
, Persona de Mediana Edad
, Proteínas Oncogénicas Virales/genética
, Valor Predictivo de las Pruebas
, ARN Mensajero/genética
, Medición de Riesgo
, Sensibilidad y Especificidad
, Adulto Joven
, Displasia del Cuello del Útero/virología
RESUMEN
BACKGROUND: Methylation of HIN-1 is associated with poor outcomes in patients with ovarian clear cell carcinoma (OCCC), which is regarded to be an aggressive, chemo-resistant histological subtype. This study aimed to evaluate whether 5-aza-2-deoxycytidine (5-aza-2-dC) can reverse methylation of the HIN-1 gene to restore chemo-sensitivity of OCCC and the possible mechanism. METHODS: In vitro flow cytometric analysis and evaluation of caspase-3/7 activity of paclitaxel-sensitive and resistant OCCC cell lines were performed. Methylation status and expression changes of HIN-1 in the OCCC cell lines treated with 5-aza-2-dC were evaluated, and immunohistochemical staining of HIN-1 in OCCC tissues was performed. In vivo tumor growth with or without 5-aza-2-dC treatment was analyzed, and Western blotting of AKT-mTOR signaling-related molecules was performed. RESULTS: G2-M phase arrest was absent in paclitaxel-resistant OCCC cells after treatment with the cytotoxic drug. The caspase activities of the chemo-resistant OCCC cells were lower than those of the chemo-sensitive OCCC cells when treated with paclitaxel. Methylation of HIN-1 was noted in paclitaxel-resistant OCCC cell lines and cancerous tissues. 5-aza-2-dC reversed the methylation of HIN-1, re-activated the expression of HIN-1, and then suppressed the in vivo tumor growth of paclitaxel-resistant OCCC cells. Immunoblotting revealed that phospho-AKT473 and phospho-mTOR were significantly increased in HIN-1-methylated paclitaxel-resistant OCCC cell lines. However, the expressions of phospho-AKT at Ser473 and Thr308 and phospho-mTOR decreased in the OCCC cells with a high expression of HIN-1. CONCLUSIONS: Demethylating agents can restore the HIN-1 expression in paclitaxel-resistant OCCC cells through the HIN-1-AKT-mTOR signaling pathway to inhibit tumor growth.
Asunto(s)
Adenocarcinoma de Células Claras/metabolismo , Citocinas/biosíntesis , Neoplasias Ováricas/metabolismo , Paclitaxel/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Serina-Treonina Quinasas TOR/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adulto , Anciano , Animales , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: To evaluate the role of surgery, radiation therapy and chemotherapy in the management of small cell carcinoma of the uterine cervix (SCCC) through a retrospective study of Taiwanese Gynecologic Oncology Group. METHODS: We reviewed the medical records and histological files of 144 patients with FIGO stages IA-IIB SCCC treated in 11 main hospitals in Taiwan from 1987 to 2009. RESULTS: There were 110 patients receiving primary surgery and 34 primary radiation therapy. Most patients in each group also received chemotherapy as part of primary treatment. A lower loco-regional failure rate was observed in patients who received primary radiation therapy than in those who had primary surgery (6% vs. 27%; P=0.009). The 5-year overall survival (OS) was 89% for 13 surgically treated patients with cervical tumor ≤2cm and no lymphovascular space involvement (LVSI) in whom recurrence was noted in 2 of 4 patients without receiving adjuvant chemotherapy and none in the 9 patients who had chemotherapy. Excluding these 13 patients, primary radiation therapy with at least 5cycles of platinum-based chemotherapy (n=14, including 12 stages IB2-IIB) resulted in a 5-year OS of 78%, better than that of 46% by primary surgery (n=97, including 40 stages IB2-IIB) (P=0.046). CONCLUSIONS: None of the 9 patients with cervical tumor ≤2cm and no LVSI showed disease recurrence after primary surgery and adjuvant chemotherapy. For most patients with stages I-II, primary radiation therapy with aggressive chemotherapy was associated with better survival than surgery.
Asunto(s)
Carcinoma de Células Pequeñas/radioterapia , Carcinoma de Células Pequeñas/cirugía , Neoplasias del Cuello Uterino/radioterapia , Neoplasias del Cuello Uterino/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Pequeñas/patología , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Retrospectivos , Taiwán , Neoplasias del Cuello Uterino/patologíaRESUMEN
Recent experimental work has described an elegant pattern of branching in the development of the lung. Multiple forms of branching have been identified, including side branching and tip bifurcation. A particularly interesting feature is the phenomenon of 'orthogonal rotation of the branching plane'. The lung must fill 3D space with the essentially 2D phenomenon of branching. It accomplishes this by rotating the branching plane by 90° with each generation. The mechanisms underlying this rotation are not understood. In general, the programmes that underlie branching have been hypothetically attributed to genetic 'subroutines' under the control of a 'global master routine' to invoke particular subroutines at the proper time and location, but the mechanisms of these routines are not known. Here, we demonstrate that fundamental mechanisms, the reaction and diffusion of biochemical morphogens, can create these patterns. We used a partial differential equation model that postulates three morphogens, which we identify with specific molecules in lung development. We found that cascades of branching events, including side branching, tip splitting and orthogonal rotation of the branching plane, all emerge immediately from the model, without further assumptions. In addition, we found that one branching mode can be easily switched to another, by increasing or decreasing the values of key parameters. This shows how a 'global master routine' could work by the alteration of a single parameter. Being able to simulate cascades of branching events is necessary to understand the critical features of branching, such as orthogonal rotation of the branching plane between successive generations, and branching mode switch during lung development. Thus, our model provides a paradigm for how genes could possibly act to produce these spatial structures. Our low-dimensional model gives a qualitative understanding of how generic physiological mechanisms can produce branching phenomena, and how the system can switch from one branching pattern to another using low-dimensional 'control knobs'. The model provides a number of testable predictions, some of which have already been observed (though not explained) in experimental work.
Asunto(s)
Pulmón/embriología , Modelos Biológicos , Organogénesis , Animales , HumanosRESUMEN
The cell is a complex system involving numerous components, which may often interact in a non-linear dynamic manner. Diseases at the cellular level are thus likely to involve multiple cellular constituents and pathways. As some drugs, or drug combinations, may act synergistically on these multiple pathways, they might be more effective than the respective single target agents. Optimizing a drug mixture for a given disease in a particular patient is particularly challenging due to both the difficulty in the selection of the drug mixture components to start out with, and the all-important doses of these drugs to be applied. For n concentrations of m drugs, in principle, n(m) combinations will have to be tested. As this may lead to a costly and time-consuming investigation for each individual patient, we have developed a Feedback System Control (FSC) technique which can rapidly select the optimal drug-dose combination from the often millions of possible combinations. By testing this FSC technique in a number of experimental systems representing different disease states, we found that the response of cells to multiple drugs is well described by a low order, rather smooth, drug-mixture-input/drug-effect-output multidimensional surface. The main consequences of this are that optimal drug combinations can be found in a surprisingly small number of tests, and that translation from in vitro to in vivo is simplified. This points to the possibility of personalized optimal drug mixtures in the near future. This unexpectedly simple input-output relationship may also lead to a simple solution for handling the issue of human diversity in cancer therapeutics.
Asunto(s)
Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Modelos Biológicos , Terapia Molecular Dirigida/métodos , Preparaciones Farmacéuticas/administración & dosificación , Medicina de Precisión/métodos , Línea Celular , Humanos , Análisis de RegresiónRESUMEN
OBJECTIVE: The aim of this study was to investigate the clinical and pathological characteristics of uterine clear cell carcinoma (UCCC) and the treatment of this disease in relation to patient outcomes. METHODS: The clinicopathological data for and the management of all patients with UCCC who presented between 1991 and 2010 at 11 member hospitals of the Taiwanese Gynecologic Oncology Group (TGOG) were retrospectively reviewed. RESULTS: There were no significant differences in 5-year overall survival (OS) rates between patients with pure UCCC (n=100) and non-pure UCCC (n=53) at the same surgical stage, with OS rates of 92.6%, and 87.7% for stage I; 83.3% and 83.3% for stage II; 64.0% and 67.8% for stage III; and 16.7% and 0% for stage IV (n=1), respectively. Tumor stage and age independently influenced the OS rate of UCCC. For the patients with early stage UCCC, the adjuvant therapy modality was the only significant prognostic factor for recurrence-free survival. The patients with early stage UCCC who received adjuvant therapy had excellent 5-year recurrence-free survival and OS rates compared to those who received radiotherapy (100% vs. 74%, p=0.01; 100% vs. 72%, p=0.03). CONCLUSIONS: The 5-year survival rates of patients with pure UCCC and non-pure UCCC were similar. The prognosis for surgical staging of patients with stage I/II UCCC was encouraging. Postoperative adjuvant platinum-based chemotherapy is recommended for patients with early stage UCCC who are at a high risk of recurrence.
Asunto(s)
Adenocarcinoma de Células Claras/patología , Adenocarcinoma de Células Claras/cirugía , Neoplasias Uterinas/patología , Neoplasias Uterinas/cirugía , Adenocarcinoma de Células Claras/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , Taiwán , Resultado del Tratamiento , Neoplasias Uterinas/mortalidadRESUMEN
RATIONALE: Left-right (LR) asymmetry is ubiquitous in animal development. Cytoskeletal chirality was recently reported to specify LR asymmetry in embryogenesis, suggesting that LR asymmetry in tissue morphogenesis is coordinated by single- or multi-cell organizers. Thus, to organize LR asymmetry at multiscale levels of morphogenesis, cells with chirality must also be present in adequate numbers. However, observation of LR asymmetry is rarely reported in cultured cells. OBJECTIVES: Using cultured vascular mesenchymal cells, we tested whether LR asymmetry occurs at the single cell level and in self-organized multicellular structures. METHODS AND RESULTS: Using micropatterning, immunofluorescence revealed that adult vascular cells polarized rightward and accumulated stress fibers at an unbiased mechanical interface between adhesive and nonadhesive substrates. Green fluorescent protein transfection revealed that the cells each turned rightward at the interface, aligning into a coherent orientation at 20° relative to the interface axis at confluence. During the subsequent aggregation stage, time-lapse videomicroscopy showed that cells migrated along the same 20° angle into neighboring aggregates, resulting in a macroscale structure with LR asymmetry as parallel, diagonal stripes evenly spaced throughout the culture. Removal of substrate interface by shadow mask-plating, or inhibition of Rho kinase or nonmuscle myosin attenuated stress fiber accumulation and abrogated LR asymmetry of both single-cell polarity and multicellular coherence, suggesting that the interface triggers asymmetry via cytoskeletal mechanics. Examination of other cell types suggests that LR asymmetry is cell-type specific. CONCLUSIONS: Our results show that adult stem cells retain inherent LR asymmetry that elicits de novo macroscale tissue morphogenesis, indicating that mechanical induction is required for cellular LR specification.
Asunto(s)
Células Madre Adultas/fisiología , Vasos Sanguíneos/embriología , Polaridad Celular , Citoesqueleto/fisiología , Mesodermo/fisiología , Animales , Vasos Sanguíneos/citología , Adhesión Celular , Técnicas de Cultivo de Célula , Movimiento Celular , Simulación por Computador , Vidrio , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mesodermo/citología , Ratones , Microscopía Fluorescente , Microscopía por Video , Modelos Biológicos , Morfogénesis , Células 3T3 NIH , Análisis Numérico Asistido por Computador , Fibras de Estrés/fisiología , Propiedades de Superficie , Factores de Tiempo , Imagen de Lapso de Tiempo , TransfecciónRESUMEN
RATIONALE: Amyotrophic lateral sclerosis (ALS) poses a significant clinical challenge due to its rapid progression and limited treatment options, often leading to deadly outcomes. Looking for effective therapeutic interventions is critical to improve patient outcomes in ALS. PATIENT CONCERNS: The patient, a 75-year-old East Asian male, manifested an insidious onset of right-hand weakness advancing with dysarthria. Comprehensive Next-generation sequencing analysis identified variants in specific genes consistent with ALS diagnosis. DIAGNOSES: ALS diagnosis is based on El Escorial diagnostic criteria. INTERVENTIONS: This study introduces a novel therapeutic approach using artificial intelligence phenotypic response surface (AI-PRS) technology to customize personalized drug-dose combinations for ALS. The patient underwent a series of phases of AI-PRS-assisted trials, initially incorporating a 4-drug combination of Ibudilast, Riluzole, Tamoxifen, and Ropinirole. Biomarkers and regular clinical assessments, including nerve conduction velocity, F-wave, H-reflex, electromyography, and motor unit action potential, were monitored to comprehensively evaluate treatment efficacy. OUTCOMES: Neurophysiological assessments supported the ALS diagnosis and revealed the co-presence of diabetic polyneuropathy. Hypotension during the trial necessitated an adaptation to a 2-drug combinational trial (ibudilast and riluzole). Disease progression assessment shifted exclusively to clinical tests of muscle strength, aligning with the patient's well-being. LESSONS: The study raises the significance of personalized therapeutic strategies in ALS by AI-PRS. It also emphasizes the adaptability of interventions based on patient-specific responses. The encountered hypotension incident highlights the importance of attentive monitoring and personalized adjustments in treatment plans. The described therapy using AI-PRS, offering personalized drug-dose combinations technology is a potential approach in treating ALS. The promising outcomes warrant further evaluation in clinical trials for searching a personalized, more effective combinational treatment for ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral , Hipotensión , Humanos , Masculino , Anciano , Riluzol/uso terapéutico , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Inteligencia Artificial , Resultado del Tratamiento , Hipotensión/tratamiento farmacológicoRESUMEN
Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (< 1/10th) of the cost of next-generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer-free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus-specific methylation heterogeneity to improve upon Pap-specimen-based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high-risk patients for subsequent clinical follow-up and monitoring. The results of this study support the utility of PapDREAM as a simple, low-cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer. KEY POINTS: We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (â¼$25) for routine screening applications. Furthermore, it provides molecule-by-molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.
Asunto(s)
Metilación de ADN , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/diagnóstico , Metilación de ADN/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Persona de Mediana Edad , ADN/genética , ADN/análisis , Sensibilidad y Especificidad , Adulto , Prueba de Papanicolaou/métodos , Prueba de Papanicolaou/estadística & datos numéricosRESUMEN
We introduce a new biosensing platform for rapid protein detection that combines one of the simplest methods for biomolecular concentration, coffee ring formation, with a sensitive aptamer-based optical detection scheme. In this approach, aptamer beacons are utilized for signal transduction where a fluorescence signal is emitted in the presence of the target molecule. Signal amplification is achieved by concentrating aptamer-target complexes within liquid droplets, resulting in the formation of coffee ring "spots". Surfaces with various chemical coatings were utilized to investigate the correlation among surface hydrophobicity, concentration efficiency, and signal amplification. On the basis of our results, we found that the increase in the coffee ring diameter with larger droplet volumes is independent of surface hydrophobicity. Furthermore, we show that highly hydrophobic surfaces produce enhanced particle concentration via coffee ring formation, resulting in signal intensities 6-fold greater than those on hydrophilic surfaces. To validate this biosensing platform for the detection of clinical samples, we detected α-thrombin in human serum and 4-fold-diluted whole blood. Coffee ring spots from serum and blood produced detection signals up to 40 times larger than those from samples in liquid droplets. Additionally, this biosensor exhibits a lower limit of detection of 2 ng/mL (54 pM) in serum, and 4 ng/mL (105 pM) in blood. On the basis of its simplicity and high performance, this platform demonstrates immense potential as an inexpensive diagnostic tool for the detection of disease biomarkers, particularly for use in developing countries that lack the resources and facilities required for conventional biodetection practices.
Asunto(s)
Aptámeros de Nucleótidos/síntesis química , Técnicas Biosensibles/métodos , Poliestirenos/química , Trombina/análisis , Técnicas Biosensibles/instrumentación , Carbocianinas/química , Coloides , Colorantes Fluorescentes/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Microscopía Fluorescente , TemperaturaRESUMEN
Herpes simplex virus type 1 (HSV-1) is known to cause diseases of various severities. There is increasing interest to find drug combinations to treat HSV-1 by reducing drug resistance and cytotoxicity. Drug combinations offer potentially higher efficacy and lower individual drug dosage. In this paper, we report a new application of fractional factorial designs to investigate a biological system with HSV-1 and six antiviral drugs, namely, interferon alpha, interferon beta, interferon gamma, ribavirin, acyclovir, and tumor necrosis factor alpha. We show how the sequential use of two-level and three-level fractional factorial designs can screen for important drugs and drug interactions, as well as determine potential optimal drug dosages through the use of contour plots. Our initial experiment using a two-level fractional factorial design suggests that there is model inadequacy and that drug dosages should be reduced. A follow-up experiment using a blocked three-level fractional factorial design indicates that tumor necrosis factor alpha has little effect and that HSV-1 infection can be suppressed effectively by using the right combination of the other five antiviral drugs. These observations have practical implications in the understanding of antiviral drug mechanism that can result in better design of antiviral drug therapy.
Asunto(s)
Antivirales/farmacología , Combinación de Medicamentos , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Modelos Estadísticos , Aciclovir/farmacología , Farmacorresistencia Viral , Humanos , Interferón-alfa/farmacología , Interferón beta/farmacología , Interferón gamma/farmacología , Ribavirina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Background: Inter- and intra-individual variability in tacrolimus dose requirements mandates empirical clinician-titrated dosing that frequently results in deviation from a narrow target range. Improved methods to individually dose tacrolimus are needed. Our objective was to determine whether a quantitative, dynamically-customized, phenotypic-outcome-guided dosing method termed Phenotypic Personalized Medicine (PPM) would improve target drug trough maintenance. Methods: In a single-center, randomized, pragmatic clinical trial ( NCT03527238 ), 62 adults were screened, enrolled, and randomized prior to liver transplantation 1:1 to standard-of-care (SOC) clinician-determined or PPM-guided dosing of tacrolimus. The primary outcome measure was percent days with large (>2 ng/mL) deviation from target range from transplant to discharge. Secondary outcomes included percent days outside-of-target-range and mean area-under-the-curve (AUC) outside-of-target-range per day. Safety measures included rejection, graft failure, death, infection, nephrotoxicity, or neurotoxicity. Results: 56 (29 SOC, 27 PPM) patients completed the study. The primary outcome measure was found to be significantly different between the two groups. Patients in the SOC group had a mean of 38.4% of post-transplant days with large deviations from target range; the PPM group had 24.3% of post-transplant days with large deviations; (difference -14.1%, 95% CI: -26.7 to -1.5 %, P=0.029). No significant differences were found in the secondary outcomes. In post-hoc analysis, the SOC group had a 50% longer median length-of-stay than the PPM group [15 days (Q1-Q3: 11-20) versus 10 days (Q1-Q3: 8.5-12); difference 5 days, 95% CI: 2-8 days, P=0.0026]. Conclusions: PPM guided tacrolimus dosing leads to better drug level maintenance than SOC. The PPM approach leads to actionable dosing recommendations on a day-to-day basis. Lay Summary: In a study on 62 adults who underwent liver transplantation, researchers investigated whether a new dosing method called Phenotypic Personalized Medicine (PPM) would improve daily dosing of the immunosuppression drug tacrolimus. They found that PPM guided tacrolimus dosing leads to better drug level maintenance than the standard-of-care clinician-determined dosing. This means that the PPM approach leads to actionable dosing recommendations on a day-to-day basis and can help improve patient outcomes.
RESUMEN
In preclinical studies, a new antituberculosis drug regimen markedly reduced the time required to achieve relapse-free cure. This study aimed to preliminarily evaluate the efficacy and safety of this four-month regimen, consisting of clofazimine, prothionamide, pyrazinamide and ethambutol, with a standard six-month regimen in patients with drug-susceptible tuberculosis. An open-label pilot randomized clinical trial was conducted among the patients with newly diagnosed bacteriologically-confirmed pulmonary tuberculosis. The primary efficacy end-point was sputum culture negative conversion. Totally, 93 patients were included in the modified intention-to-treat population. The rates of sputum culture conversion were 65.2% (30/46) and 87.2% (41/47) for short-course and standard regimen group, respectively. There was no difference on two-month culture conversion rates, time to culture conversion, nor early bactericidal activity (P > 0.05). However, patients on short-course regimen were observed with lower rates of radiological improvement or recovery and sustained treatment success, which was mainly attributed to higher percent of patients permanently changed assigned regimen (32.1% vs. 12.3%, P = 0.012). The main cause for it was drug-induced hepatitis (16/17). Although lowering the dose of prothionamide was approved, the alternative option of changing assigned regimen was chosen in this study. While in per-protocol population, sputum culture conversion rates were 87.0% (20/23) and 94.4% (34/36) for the respective groups. Overall, the short-course regimen appeared to have inferior efficacy and higher incidence of hepatitis but desired efficacy in per-protocol population. It provides the first proof-of-concept in humans of the capacity of the short-course approach to identify drug regimens that can shorten the treatment time for tuberculosis.
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Clofazimina , Tuberculosis , Humanos , Clofazimina/efectos adversos , Protionamida , Quimioterapia Combinada , Antituberculosos/efectos adversos , Tuberculosis/tratamiento farmacológico , Pirazinamida/efectos adversos , Resultado del Tratamiento , IsoniazidaRESUMEN
BACKGROUND: This study is to analyze promoter methylation of various tumor suppressor genes in different types of ovarian carcinoma and to identify potential therapeutic targets of ovarian clear cell adenocarcinoma (OCCA). MATERIALS AND METHODS: The promoter methylation statuses of 40 genes in primary ovarian carcinomas including 47 clear- and 63 non-clear-cell type tissues, 6 OCCA cell lines, 29 benign ovarian endometriotic cysts, and 31 normal controls were analyzed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The MS-MLPA results were correlated with clinicopathological features and outcomes of 47 OCCA patients. Functions of the target genes were further explored by Western Blot Analysis, apoptosis assay, and caspase-3/7 activity analysis. RESULTS: Frequencies of methylated RASSF1A, CDH13, CACNA1A, HIN-1, and sFRP5 genes in OCCA tissues were significantly higher than those in non-OCCA cancerous tissues and benign endometriotic cysts. The expected OS for patients with methylated promoters of HIN-1 was significantly worse than those for patients without methylated HIN-1 (30% vs. 62%, p = 0.002). The HIN-1 gene was over-expressed in ES2 cells, a significant reduction in cell growth and induction of apoptosis, and increasing paclitaxel sensitivity by reducing phosphorylation of Akt were observed. CONCLUSIONS: Methylation of HIN-1 promoter is a novel epigenetic biomarker associated with poor outcomes in OCCA patients. Ectopic expression of the HIN-1 gene increased paclitaxel sensitivity which is partly through Akt pathway.
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Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/mortalidad , Citocinas/genética , Metilación de ADN , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Adenocarcinoma de Células Claras/patología , Adulto , Anciano , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Paclitaxel/farmacología , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
Kidney transplant recipients who have abnormally high creatinine levels in their blood often have allograft dysfunction secondary to rejection. Creatinine has become the preferred marker for renal dysfunction and is readily available in hospital clinical settings. We developed a rapid and accurate polymer-based electrochemical point-of-care (POC) assay for creatinine detection from whole blood to identify allograft dysfunction. The creatinine concentrations of 19 blood samples from transplant recipients were measured directly from clinical serum samples by the conducting polymer-based electrochemical (EC) sensor arrays. These measurements were compared to the traditional clinical laboratory assay. The time required for detection was <5 min from sample loading. Sensitivity of the detection was found to be 0.46 mg/dL of creatinine with only 40 µL sample in the creatinine concentration range of 0 mg/dL to 11.33 mg/dL. Signal levels that were detected electrochemically correlated closely with the creatinine blood concentration detected by the UCLA Ronald Reagan Medical Center traditional clinical laboratory assay (correlation coefficient = 0.94). This work is encouraging for the development of a rapid and accurate POC device for measuring creatinine levels in whole blood.
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Creatinina/sangre , Técnicas Electroquímicas , Riñón/fisiopatología , Polímeros/química , Anticuerpos/inmunología , Técnicas Biosensibles , Humanos , Trasplante de Riñón , Sistemas de Atención de Punto , Trasplante HomólogoRESUMEN
BACKGROUND: At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA). METHODS: Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium. RESULTS: The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44(high), CD24(low), and AC133(+). These cells also demonstrated high BMP-2, BMP4, TGF-ß, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α2ß1, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α2ß1, and CD146 surface marker expression than the epithelial type cells. CONCLUSION: The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.