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BACKGROUND: Effective peritoneal dialysis (PD) training is essential for performing dialysis at home and reducing the risk of peritonitis and other PD-related infections. Virtual reality (VR) is an innovative learning tool that is able to combine theoretical information, interactivity, and behavioral instructions while offering a playful learning environment. To improve patient training for PD, Fresenius Medical Care launched the stayâ¢safe MyTraining VR, a novel educational program based on the use of a VR headset and a handheld controller. OBJECTIVE: This qualitative assessment aims to investigate opinions toward the new tool among the health care professionals (HCPs) who were responsible for implementing the VR application. METHODS: We recruited nursing staff and nephrologists who have gained practical experience with the stayâ¢safe MyTraining VR within pilot dialysis centers. Predetermined open-ended questions were administered during individual and group video interviews. RESULTS: We interviewed 7 HCPs who have 2 to 20 years of experience in PD training. The number of patients trained with the stayâ¢safe MyTraining VR ranged from 2 to 5 for each professional. The stayâ¢safe MyTraining VR was well accepted and perceived as a valuable supplementary tool for PD training. From the respondents' perspective, the technology improved patients' learning experience by facilitating the internalization of both medical information and procedural skills. HCPs highlighted that the opportunity offered by VR to reiterate training activities in a positive and safe learning environment, according to each patient's needs, can facilitate error correction and implement a standardized training curriculum. However, VR had limited use in the final phase of the patient PD training program, where learners need to get familiar with the handling of the materials. Moreover, the traditional PD training was still considered essential to manage the emotional and motivational aspects and address any patient-specific application-oriented questions. In addition to its use within PD training, VR was perceived as a useful tool to support the decision-making process of patients and train other HCPs. Moreover, VR introduction was associated with increased efficiency and productivity of HCPs because it enabled them to perform other activities while the patient was practicing with the device. As for patients' acceptance of the new tool, interviewees reported positive feedback, including that of older adults. Limited use with patients experiencing dementia or severe visual impairment or lacking sensomotoric competence was mentioned. CONCLUSIONS: The stayâ¢safe MyTraining VR is suggested to improve training efficiency and efficacy and thus could have a positive impact in the PD training scenario. Our study offers a process proposal that can serve as a guide to the implementation of a VR-based PD training program within other dialysis centers. Dedicated research is needed to assess the operational benefits and the consequences on patient management.
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Personal de Salud , Diálisis Peritoneal , Investigación Cualitativa , Realidad Virtual , Humanos , Diálisis Peritoneal/métodos , Personal de Salud/educación , Femenino , Masculino , Adulto , Educación del Paciente como Asunto/métodos , Persona de Mediana EdadRESUMEN
Background and Objectives: Cardiovascular (CV) disease is the main cause of morbidity and mortality in patients suffering from chronic kidney disease (CKD). Although it is widely recognized that CV risk assessment represents an essential prerequisite for clinical management, existing prognostic models appear not to be entirely adequate for CKD patients. We derived a literature-based, naïve-bayes model predicting the yearly risk of CV hospitalizations among patients suffering from CKD, referred as the CArdiovascular, LIterature-Based, Risk Algorithm (CALIBRA). Methods: CALIBRA incorporates 31 variables including traditional and CKD-specific risk factors. It was validated in two independent CKD populations: the FMC NephroCare cohort (European Clinical Database, EuCliD®) and the German Chronic Kidney Disease (GCKD) study prospective cohort. CALIBRA performance was evaluated by c-statistics and calibration charts. In addition, CALIBRA discrimination was compared with that of three validated tools currently used for CV prediction in CKD, namely the Framingham Heart Study (FHS) risk score, the atherosclerotic cardiovascular disease risk score (ASCVD), and the Individual Data Analysis of Antihypertensive Intervention Trials (INDANA) calculator. Superiority was defined as a ΔAUC>0.05. Results: CALIBRA showed good discrimination in both the EuCliD® medical registry (AUC 0.79, 95%CI 0.76-0.81) and the GCKD cohort (AUC 0.73, 95%CI 0.70-0.76). CALIBRA demonstrated improved accuracy compared to the benchmark models in EuCliD® (FHS: ΔAUC=-0.22, p<0.001; ASCVD: ΔAUC=-0.17, p<0.001; INDANA: ΔAUC=-0.14, p<0.001) and GCKD (FHS: ΔAUC=-0.16, p<0.001; ASCVD: ΔAUC=-0.12, p<0.001; INDANA: ΔAUC=-0.04, p<0.001) populations. Accuracy of the CALIBRA score was stable also for patients showing missing variables. Conclusion: CALIBRA provides accurate and robust stratification of CKD patients according to CV risk and allows score calculations with improved accuracy compared to established CV risk scores also in real-world clinical cohorts with considerable missingness rates. Our results support the generalizability of CALIBRA across different CKD populations and clinical settings.
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Current equation-based risk stratification algorithms for kidney failure (KF) may have limited applicability in real world settings, where missing information may impede their computation for a large share of patients, hampering one from taking full advantage of the wealth of information collected in electronic health records. To overcome such limitations, we trained and validated the Prognostic Reasoning System for Chronic Kidney Disease (PROGRES-CKD), a novel algorithm predicting end-stage kidney disease (ESKD). PROGRES-CKD is a naïve Bayes classifier predicting ESKD onset within 6 and 24 months in adult, stage 3-to-5 CKD patients. PROGRES-CKD trained on 17,775 CKD patients treated in the Fresenius Medical Care (FMC) NephroCare network. The algorithm was validated in a second independent FMC cohort (n = 6760) and in the German Chronic Kidney Disease (GCKD) study cohort (n = 4058). We contrasted PROGRES-CKD accuracy against the performance of the Kidney Failure Risk Equation (KFRE). Discrimination accuracy in the validation cohorts was excellent for both short-term (stage 4-5 CKD, FMC: AUC = 0.90, 95%CI 0.88-0.91; GCKD: AUC = 0.91, 95% CI 0.86-0.97) and long-term (stage 3-5 CKD, FMC: AUC = 0.85, 95%CI 0.83-0.88; GCKD: AUC = 0.85, 95%CI 0.83-0.88) forecasting horizons. The performance of PROGRES-CKD was non-inferior to KFRE for the 24-month horizon and proved more accurate for the 6-month horizon forecast in both validation cohorts. In the real world setting captured in the FMC validation cohort, PROGRES-CKD was computable for all patients, whereas KFRE could be computed for complete cases only (i.e., 30% and 16% of the cohort in 6- and 24-month horizons). PROGRES-CKD accurately predicts KF onset among CKD patients. Contrary to equation-based scores, PROGRES-CKD extends to patients with incomplete data and allows explicit assessment of prediction robustness in case of missing values. PROGRES-CKD may efficiently assist physicians' prognostic reasoning in real-life applications.
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Fallo Renal Crónico , Insuficiencia Renal Crónica , Insuficiencia Renal , Algoritmos , Teorema de Bayes , Progresión de la Enfermedad , Humanos , Fallo Renal Crónico/diagnóstico , Pronóstico , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/epidemiología , Medición de RiesgoRESUMEN
BACKGROUND: This study aimed to determine the costs and distribution of healthcare spending of patients with chronic kidney disease (CKD) at stages 3 and 4 and on dialysis both at the individual and population level in Germany. METHODS: The study took the perspective of the German statutory health insurance (SHI) system and analyzed claims data on 3,687,015 insurees from the year 2014. To extrapolate costs to the whole SHI population, a literature search on the prevalence of CKD was conducted. RESULTS: Average costs per person per year in an age- and gender-matched control group of the normal population were 2,876 (95% confidence interval [CI], 2,798 to 2,955) and ≥2.8-fold higher in CKD patients (8,030 [95% CI, 7,848 to 8,212] at CKD stage 3, 9,760 [95% CI, 9,266 to 10,255] at CKD stage 4, and 44,374 [95% CI, 43,608 to 45,139] on dialysis). At CKD stages 3 and 4 the major cost driver was hospitalizations, contributing to more than 50% of total expenditures. Among dialysis patients, hospitalizations and dialysis-treatment costs contributed to 23% and 53% of total healthcare spending, respectively. At CKD stages 3 and 4, patients with the highest 20% of healthcare spending showed a considerable increase in per-patient costs over the reference population, while the bottom 80% of patients generated only moderately higher per-patient costs (p < 0.001). Comparing total CKD costs to total SHI expenditures yields that 10.2% of SHI expenditures was driven by patients at CKD stages 3 and 4 and 1.6% by dialysis patients. CONCLUSIONS: Healthcare spending of patients with CKD at stages 3 and 4 and on dialysis is concentrated among a small number of high-need patients. As hospitalizations and dialysis treatment are key drivers of total expenditures, strategies that lead to a reduction in hospitalizations, delay in dialysis onset, or increase in the availability of kidney donors should become important considerations by policymakers.
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Costos y Análisis de Costo , Insuficiencia Renal Crónica/economía , Anciano , Bases de Datos Factuales , Femenino , Alemania , Hospitalización/economía , Humanos , Revisión de Utilización de Seguros , Masculino , Diálisis Renal/economía , Insuficiencia Renal Crónica/patología , Índice de Severidad de la EnfermedadRESUMEN
Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.
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Carbón Orgánico/química , Cresoles/química , Indicán/química , Fenilacetatos/química , Povidona/química , Insuficiencia Renal/sangre , Ésteres del Ácido Sulfúrico/química , Uremia , Adsorción , Recuento de Células Sanguíneas , Humanos , Diálisis RenalRESUMEN
While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.
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Proteínas de Ciclo Celular/genética , Replicación del ADN , Microcefalia/genética , Microcefalia/patología , Mutación , Proteínas Nucleares/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/patología , Transcriptoma , Animales , Mapeo Cromosómico , Femenino , Ligamiento Genético , Inestabilidad Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Noqueados , Microcefalia/etiología , Osteocondrodisplasias/etiología , Linaje , Embarazo , Empalme del ARN , Análisis de Secuencia de ARN , Secuenciación Completa del GenomaRESUMEN
PURPOSE: Chronic itch (CI) is a common symptom in haemodialysis (HD) patients affecting about every 4th HD patient. Though it is known that health-related quality of life (HRQOL) is impaired in HD patients, data are lacking on how CI influences HRQOL in this group of patients, also regarding sex differences and the role of sleep and pain. METHODS: In order to investigate the itch prevalence and to investigate associated factors, GEHIS (German Epidemiology Haemodialysis Itch Study) was established as first cross-sectional study being representative for all dialysis units in Germany by considering their regional distribution and size of the located city according to population. HRQOL of 860 HD patients from a randomly selected cluster sample of 25 dialysis units was investigated using patient-reported outcomes as the Short-Form Health Survey (SF-12), the Hospital Anxiety and Depression Scale (HADS) as well as sleep and pain. Additionally, itch-specific QOL was assessed in HD patients affected by CI using the validated ItchyQoL. RESULTS: 42.8 % of HD patients were female. The mean age was 67.2 years (SD ± 13.5). Female patients showed significantly worse values in the mental component summary of the SF-12 as well as in the HADS subscale anxiety compared to male patients. Itch-specific HRQOL did not subject to sex. Patients with impairments in quality of sleep showed significantly worse values in both the SF-12 and the HADS subscales, however not in the ItchyQoL. The occurrence of pain was significantly associated with impaired SF-12, HADS and the ItchyQoL. Multivariate linear regression showed the itch-specific HRQOL to be significantly impaired with rising severity of CI. CONCLUSIONS: Impairments in HRQOL are a serious problem in HD patients. CI means an additional burden in affected patients, also impairing their HRQOL. GEHIS showed significant sex differences in HRQOL outcomes also in HD patients. CI and pain as well as sleep frequently occur combined and significantly impair HRQOL in HD patients. The care of HD patients should be complemented by psychosocial support and mental health services.
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Prurito/psicología , Diálisis Renal/psicología , Perfil de Impacto de Enfermedad , Anciano , Estudios Transversales , Femenino , Alemania/epidemiología , Humanos , Masculino , Prurito/etiología , Diálisis Renal/efectos adversosRESUMEN
Administration of bone marrow-derived mononuclear cells (BMC) may increase cardiac function after myocardial ischemia. However, the functional capacity of BMC derived from chronic heart failure (CHF) patients is significantly impaired. As modulation of the energy metabolism allows cells to match the divergent demands of the environment, we examined the regulation of energy metabolism in BMC from patients and healthy controls (HC). The glycolytic capacity of CHF-derived BMC is reduced compared to HC, whereas BMC of metabolically activated bone marrow after acute myocardial infarction reveal increased metabolism. The correlation of metabolic pathways with the functional activity of cells indicates an influence of metabolism on cell function. Reducing glycolysis without profoundly affecting ATP-production reversibly reduces invasion as well as colony forming capacity and abolishes proliferation of CD34(+) CD38(-) lin(-) hematopoietic stem and progenitor cells (HSPC). Ex vivo inhibition of glycolysis further reduced the pro-angiogenic activity of transplanted cells in a hind limb ischemia model in vivo. In contrast, inhibition of respiration, without affecting total ATP production, leads to a compensatory increase in glycolytic capacity correlating with increased colony forming capacity. Isolated CD34(+) , CXCR4(+) , and CD14(+) cells showed higher glycolytic activity compared to their negative counterparts. Metabolic activity was profoundly modulated by the composition of media used to store or culture BMC. This study provides first evidence that metabolic alterations influence the functional activity of human HSPC and BMC independent of ATP production. Changing the balance between respiration and glycolysis might be useful to improve patient-derived cells for clinical cardiac cell therapy. Stem Cells 2016;34:2236-2248.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Insuficiencia Cardíaca/terapia , Isquemia Miocárdica/terapia , Animales , Respiración de la Célula , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Glucólisis , Insuficiencia Cardíaca/patología , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Humanos , Metabolómica , Ratones Desnudos , MicroARNs/metabolismo , Isquemia Miocárdica/patología , Neovascularización Fisiológica , Factor de Transcripción STAT5/metabolismoRESUMEN
BACKGROUND: A representative cross-sectional study showed that chronic itch (lasting for a minimum of 6 weeks) affects 25.2 % (point prevalence) of hemodialysis (HD) patients. Pathophysiology and etiology of chronic itch (CI) in HD are still unclear. METHODS: We investigated 860 HD patients from a representative randomly selected cluster-sample considering the regional distributions of dialysis units in Germany. The current analyses report comorbidities, laboratory values and dialysis characteristics of HD patients in relation to CI. RESULTS: Diabetes was the only comorbidity that was associated with the occurrence of itch but interestingly with less CI. Except for creatinine, phosphorus, and parathormone, there were no significant associations between the occurrence and characteristics of CI and any laboratory value. Kt/V was not associated with the presence of CI. Patients dialyzed with polyarylethersulfone-membrane showed significantly more CI in all prevalence estimates and those dialyzed with polysulfone-membrane were significantly less affected by CI. CONCLUSIONS: Long-term follow-up studies will show if the type of dialysis membrane influences the development of CI in HD patients. It is most likely that several factors e.g. elevated parathormone, origin of end stage renal disease (ESRD), type of dialysis membrane, and a neuropathic component all contribute to the occurrence of CI in HD patients. Future research should consider a multifactorial origin of itch in HD.
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Prurito/diagnóstico , Prurito/epidemiología , Diálisis Renal/estadística & datos numéricos , Insuficiencia Renal Crónica/epidemiología , Insuficiencia Renal Crónica/terapia , Anciano , Causalidad , Enfermedad Crónica , Comorbilidad , Estudios Transversales , Complicaciones de la Diabetes , Femenino , Alemania/epidemiología , Humanos , Incidencia , Masculino , Prurito/sangre , Insuficiencia Renal Crónica/diagnóstico , Factores de RiesgoRESUMEN
Chronic itch is a common symptom in haemodialysis (HD) patients, which is often underestimated. The aim of this cross-sectional study was to investigate the prevalence and factors associated with chronic itch in HD patients. A total of 860 HD patients from a randomly selected cluster-sample of patients attending dialysis units in Germany were included. The patients' mean?±?SD age was 67.2?±?13.5 years, 57.2% were male. The point prevalence of chronic itch was 25.2% (95% CI 22.4-28.1), 12-month prevalence was 27.2% (95% CI 24.1-30.3) and lifetime prevalence was 35.2% (95% CI 31.9-38.3). Chronic itch was significantly less prevalent in patients with secondary glomerulonephritis as primary renal disease. A history of dry skin, eczema, and age 70 years were significantly associated with chronic itch. General health status and quality of life were significantly more impaired in subjects with chronic itch. This is the first representative cross-sectional study using a precise definition of chronic itch and using different prevalence estimates of chronic itch in HD patients. It demonstrates that chronic itch is a long-lasting burden significantly impairing patients' health.
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Prurito/epidemiología , Diálisis Renal/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Costo de Enfermedad , Estudios Transversales , Emociones , Femenino , Alemania/epidemiología , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Prospectivos , Prurito/diagnóstico , Prurito/fisiopatología , Prurito/psicología , Calidad de Vida , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Sueño , Encuestas y Cuestionarios , Factores de Tiempo , Adulto JovenRESUMEN
AIM: The aim of this paper is to develop a simulation model that analyzes cost-offsets of a hypothetical disease management program (DMP) for patients with chronic kidney disease (CKD) in Germany compared to no such program. METHODS: A lifetime Markov model with simulated 65-year-old patients with CKD was developed using published data on costs and health status and simulating the progression to end-stage renal disease (ESRD), cardiovascular disease and death. A statutory health insurance perspective was adopted. RESULTS: This modeling study shows considerable potential for cost-offsets from a DMP for patients with CKD. The potential for cost-offsets increases with relative risk reduction by the DMP and baseline glomerular filtration rate. Results are most sensitive to the cost of dialysis treatment. CONCLUSION: This paper presents a general 'prototype' simulation model for the prevention of ESRD. The model allows for further modification and adaptation in future applications.
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Cadenas de Markov , Modelos Económicos , Insuficiencia Renal Crónica/terapia , Anciano , Progresión de la Enfermedad , Alemania , Tasa de Filtración Glomerular , Humanos , Seguro de Salud/economía , Fallo Renal Crónico/economía , Fallo Renal Crónico/prevención & control , Diálisis Renal/economía , Insuficiencia Renal Crónica/economía , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
Signal-induced transcript isoform variation (TIV) includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.
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Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Exones , Variación Genética , ARN Mensajero/genética , Transcriptoma , Empalme Alternativo , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Poliadenilación , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
The EGFR-driven cell-cycle pathway has been extensively studied due to its pivotal role in breast cancer proliferation and pathogenesis. Although several studies reported regulation of individual pathway components by microRNAs (miRNAs), little is known about how miRNAs coordinate the EGFR protein network on a global miRNA (miRNome) level. Here, we combined a large-scale miRNA screening approach with a high-throughput proteomic readout and network-based data analysis to identify which miRNAs are involved, and to uncover potential regulatory patterns. Our results indicated that the regulation of proteins by miRNAs is dominated by the nucleotide matching mechanism between seed sequences of the miRNAs and 3'-UTR of target genes. Furthermore, the novel network-analysis methodology we developed implied the existence of consistent intrinsic regulatory patterns where miRNAs simultaneously co-regulate several proteins acting in the same functional module. Finally, our approach led us to identify and validate three miRNAs (miR-124, miR-147 and miR-193a-3p) as novel tumor suppressors that co-target EGFR-driven cell-cycle network proteins and inhibit cell-cycle progression and proliferation in breast cancer.
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Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Genes erbB-1/fisiología , MicroARNs/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Ensayos Analíticos de Alto Rendimiento , Humanos , Redes y Vías Metabólicas/genética , MicroARNs/fisiología , Modelos Biológicos , Unión Proteica/genética , Proteómica/métodos , Transcriptoma/genética , Transcriptoma/fisiología , Células Tumorales CultivadasRESUMEN
Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.
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Ensayos Analíticos de Alto Rendimiento/métodos , Mitosis , Fosfotransferasas/metabolismo , Interferencia de ARN , Transducción de Señal , Proliferación Celular/efectos de los fármacos , Pruebas de Enzimas , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Fenotipo , Fosfotransferasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Factores de TiempoRESUMEN
The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of the indicator cells, giving rise to a strong fluorescence signal. Furthermore, the authors show that the nonspecific cytotoxic compound sodium azide kills both cancer and noncancer cells, and no fluorescence signal is obtained. Hence, this assay system favors the selection of compounds that specifically target cancer cells and decreases the probability of selecting nonspecific cytotoxic molecules. Z factors of up to 0.85 were obtained, indicating an excellent assay that can be used for high-throughput screening.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Técnicas de Cocultivo , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Neoplasias Renales/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Fluorescencia , Humanos , Indoles/farmacología , Neoplasias Renales/patología , Especificidad de Órganos , Quinazolinas , Reproducibilidad de los Resultados , Azida Sódica/efectos adversos , Sulfonamidas/farmacología , Tirfostinos/farmacologíaRESUMEN
BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.
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Colon/patología , Productos del Gen nef/química , Activación de Linfocitos , Mutación , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Colon/virología , Productos del Gen nef/genética , Productos del Gen nef/metabolismo , Humanos , Linfopenia/virología , Macaca nemestrina , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/patología , Enfermedades de los Monos/virología , Fenotipo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/metabolismo , Viremia/virología , Replicación ViralRESUMEN
Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34(+) hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source. When cytokine-mobilized peripheral blood CD34(+) cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells were readily transduced with lentiviral vectors when exposed to vector particles between 48 and 72 hours. Among the genetic elements that may enhance fetal hemoglobin production is an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal gamma-globin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of relatively weak erythroid-specific promoters induced significant amounts of HbF (up to 20%) in erythroblasts derived from adult CD34(+) cells without altering their capacity for erythroid maturation and only modestly reducing the total numbers of cells that accumulate in culture after transduction. These observations demonstrate the potential for sequence-specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia.
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Eritroblastos/metabolismo , Hemoglobina Fetal/biosíntesis , Regiones Promotoras Genéticas/genética , Transactivadores/metabolismo , Dedos de Zinc , gamma-Globinas/genética , Adulto , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromatina/metabolismo , Citocinas/farmacología , Eritroblastos/citología , Eritroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Movilización de Célula Madre Hematopoyética , Humanos , Lentivirus/genética , Modelos Genéticos , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Unión Proteica/efectos de los fármacos , Transducción GenéticaRESUMEN
Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TF(ZF)s). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TF(ZF) encoding gene. Using this strategy, we successfully selected specific TF(ZF)s that upregulate the expression of the gamma-globin promoter. Selected transcription factors induced the expression of gamma-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This new retroviral approach might be used to select other TF(ZF)s but might also be generalized for the selection of other protein and small-molecule interactions.
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Retroviridae/genética , Retroviridae/fisiología , Factores de Transcripción/metabolismo , Animales , Línea Celular , Humanos , Ratones , Modelos Teóricos , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Dedos de Zinc , gamma-Globinas/genéticaRESUMEN
RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.
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ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Expresión Génica , Plásmidos/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuencia de Bases , Doxiciclina/farmacología , Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , ARN/biosíntesis , ARN/genética , ARN Catalítico/metabolismoRESUMEN
The major T cell growth factor interleukin-2 (IL-2) is secreted by activated T cells in response to antigenic stimulation. This requires signal transduction via the CD3/TCR complex and the CD28 coreceptor, leading to activation of mitogen-activated protein kinase (MAPK) and calcineurin/NF-AT signaling pathways. We observed that simian immunodeficiency virus derived from African green monkeys (SIVagm3) is a potent activator of IL-2 gene expression. IL-2 promoter studies in A3.01 T cells demonstrated that SIVagm3 induced an up to 38-fold increased transcriptional activation of the IL-2 promoter. Inhibition of MAPK signaling pathways using inhibitors of MEK, JNK or p38 abolished SIVagm3-induced IL-2 activation in a dose-dependent manner. In contrast, the immunosuppressive drug cyclosporin A (CyA), a classical IL-2 inhibitor that blocks calcineurin activity, had no effect. Consistent with this finding, the nuclear factor of activated T cells (NF-AT), which is activated by calcineurin, was not induced by SIVagm3. Analyzing further transcription factor binding sites located on the IL-2 promoter we found that SIVagm3 did mainly promote transcriptional activation of the CD28/AP-1 and NF-kappaB responsive elements. These DNA elements were also induced by the viral transactivator protein (Tat) and expression of Tat was sufficient to activate IL-2 induction in stimulated cells. Our results show that SIVagm3 is capable of stimulating IL-2 gene expression via molecular mechanisms different from those induced during classical T cell activation.