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OBJECTIVES: To systematically assess the efficacy of percutaneous ultrasound-guided needle tenotomy (PUNT) in the treatment of chronic tendinopathy and fasciopathy. METHODS: A comprehensive literature search was performed with the following search terms: tendinopathy, tenotomy, needling, Tenex, fasciotomy, ultrasound-guided, and percutaneous. Inclusion criteria consisted of original studies evaluating pain or function improvement after PUNT. Meta-analyses investigating standard mean differences were performed to assess the pain and function improvement. RESULTS: Thirty-five studies with 1674 participants (1876 tendons) were enrolled in this article. Of which 29 articles were included in meta-analysis and the remaining 9 articles without enough numeric data were included in descriptive analysis. PUNT significantly alleviated pain with the standard mean difference of 2.5 (95% CI: 2.0-3.0; p < 0.05), 2.2 (95% confidence interval (CI): 1.8-2.7; p < 0.05), and 3.6 (95% CI: 2.8-4.5; p < 0.05) points in short-term, intermediate-term, and long-term follow-up intervals, respectively. It was also associated with marked improvement in function with 1.4 (95% CI: 1.1-1.8; p < 0.05), 1.8 (95% CI: 1.3-2.2; p < 0.05), and 2.1 (95% CI: 1.6-2.6; p < 0.05) points, respectively in short-term, intermediate-term, and long-term follow-ups. CONCLUSION: PUNT improved pain and function at short-term intervals with persistent results on intermediate- and long-term follow-ups. PUNT can be considered an appropriate minimally invasive treatment for chronic tendinopathy with a low rate of complications and failures. CLINICAL RELEVANCE: Tendinopathy and fasciopathy are two common musculoskeletal complaints that can cause prolonged pain and disability. PUNT as a treatment option could improve pain intensity and function. KEY POINTS: ⢠The best improvement in pain and function was achieved after the first 3 months following PUNT and was continued to the intermediate- and long-term follow-ups. ⢠No significant difference was found between different tenotomy methods in terms of pain and function improvement. ⢠PUNT is a minimally invasive procedure with promising results and low complication rates for treatments of chronic tendinopathy.
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Tendinopatía , Tenotomía , Humanos , Tenotomía/métodos , Tendones/diagnóstico por imagen , Tendones/cirugía , Tendinopatía/diagnóstico por imagen , Tendinopatía/cirugía , Dolor , Ultrasonografía Intervencional/métodosRESUMEN
Future distribution grids will be subjected to fluctuations in voltages and power flows due to the presence of renewable sources with intermittent power generation. The advanced smart metering infrastructure (AMI) enables the distribution system operators (DSOs) to measure and analyze electrical quantities such as voltages, currents and power at each customer connection point. Various smart grid applications can make use of the AMI data either in offline or close to real-time mode to assess the grid voltage conditions and estimate losses in the lines/cables. The outputs of these applications can enable DSOs to take corrective action and make a proper plan for grid upgrades. In this paper, the process of development and deployment of applications for improving the observability of distributions grids is described, which consists of the novel deployment framework that encompasses the proposition of data collection, communication to the servers, data storage, and data visualization. This paper discussed the development of two observability applications for grid monitoring and loss calculation, their validation in a laboratory setup, and their field deployment. A representative distribution grid in Denmark is chosen for the study using an OPAL-RT real-time simulator. The results of the experimental studies show that the proposed applications have high accuracy in estimating grid voltage magnitudes and active energy losses. Further, the field deployment of the applications prove that DSOs can gain insightful information about their grids and use them for planning purposes.
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Sistemas de Computación , Electricidad , Almacenamiento y Recuperación de la InformaciónRESUMEN
PURPOSE: Definition of targets in multiparametric MRI (mpMRI) prior to MRI/TRUS fusion prostate biopsy either by urologist or radiologist, as a prose report or by illustration is crucial for accurate targeted biopsies (TB). The objective was to analyze the effect of MRI reporting on target definition and cancer detection. METHODS: 202 patients underwent MRI/TRUS fusion biopsy with Artemis™ (Eigen, USA). mpMRI results were submitted in written form to urologists, who marked the targets in the proprietary software. An expert uroradiologist reviewed and marked mpMRI targets blinded to biopsy data. We compared number, localization and volume of targets between the observers and analyzed whether variations impaired TB results by bivariate and logistic regression models. RESULTS: Interobserver variability was moderate regarding number and low regarding localization of targets. Urologists overestimated target volumes significantly compared to radiologists (p = 0.045) and matching target volume between both observers was only 43.9%. Overall cancer detection rate was 69.8 and 52.0% by TB. A higher matching target volume was a significant predictor of cancer in TB (p < 0.001). Logistic regression revealed prostate volume and PI-RADS as independent predictors. Defining targets in incorrect T2w slices in the cranio-caudal axis are one presumable reason for missing cancer in TB. CONCLUSIONS: A high concordance of the target definition between radiologist and urologist is mandatory for accurate TB. Optimized ROI definition is recommended to improve TB results, preferably as contouring in MRI sequences by the radiologist or, if not feasible, by precise MRI reports including specific localization in sequence and slice as well as an illustration. High prostate volume and low PI-RADS score have to be considered as limiting factors for target definition.
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Biopsia Guiada por Imagen/métodos , Imagen por Resonancia Magnética Intervencional , Próstata/patología , Ultrasonografía Intervencional , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Neoplasias de la Próstata/patología , Radiología Intervencionista , Recto , Programas Informáticos , UrologíaRESUMEN
Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrP(C)). They propagate by recruiting host-encoded PrP(C) although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrP(C) expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrP(C) deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state.
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Diferenciación Celular/genética , Matriz Extracelular/metabolismo , Redes Reguladoras de Genes/genética , Modelos Moleculares , Proteínas PrPC/metabolismo , Priones/genética , Transcriptoma/genética , Animales , Clonación Molecular , Citometría de Flujo , Humanos , Ratones , Análisis por Micromatrices , Microscopía Fluorescente , Proteínas PrPC/genética , Priones/química , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrofotometría , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
The treatment of endoleaks type II had to be adapted to the anatomy of each individual patient. The laser-assisted perforation of the prosthesis can be an easier method to reach the aneurysm sac directly than using transarterial or translumbar approaches.
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Aneurisma de la Aorta Abdominal/cirugía , Endofuga/cirugía , Hemostáticos/administración & dosificación , Complicaciones Posoperatorias/cirugía , Trombina/administración & dosificación , Anciano , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Implantación de Prótesis Vascular , Embolización Terapéutica , Humanos , Inyecciones Intravenosas , Coagulación con Láser , Masculino , Stents , Resultado del TratamientoRESUMEN
OBJECTIVE: To evaluate the role of interstitial pulsed dose rate brachytherapy (PDR-BT) in multimodality treatment of locally advanced primary or recurrent rectal and sigmoid cancer with high risk of microscopic incomplete resection (R1). METHODS AND MATERIAL: A total of 73 consecutive patients (recurrent/primary: 40/33) were treated with PDR-BT between 2001 and 2010. Patients received preoperative external beam radiotherapy (EBRT) and concomitant chemotherapy. Following resection of the tumor and the involved pelvic organs, a median of four (3-8) catheters were sutured to the tumor bed with a distance of approximately 1 cm between the catheters. A target respecting the catheters with a margin of 5 mm was contoured on computed tomography (CT) and three-dimensional (3D) dose planning with a planning aim for BT of D90 > 30 Gy, (0.6 Gy/pulse, 1 pulse/h) was performed. Previously irradiated patients (27%) underwent surgery that was directly followed by PDR-BT. Postoperative EBRT was then applied to the tumor bed 3-5 weeks after PDR-BT. RESULTS: A total of 23 patients (31%) received a radical resection (R0) and 45 patients (62%) received an R1 resection. Five patients (7%) received a macroscopic incomplete resection (R2). The five-year overall survival was 33%. Local control at five years was 67% for patients who received a R0 resection and 32% for patients who received an R1 resection. The five-year actuarial risk of a grade 3-4 BT-related complication was 5%. CONCLUSIONS: Meaningful disease control and survival can be obtained at an acceptable rate of late morbidity in selected patients with locally advanced primary and recurrent rectal or sigmoid cancer using (chemo) RT, extensive surgery and PDR-BT when a high risk of an R1 resection is expected.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Braquiterapia/mortalidad , Recurrencia Local de Neoplasia/terapia , Neoplasias del Recto/terapia , Neoplasias del Colon Sigmoide/terapia , Adulto , Anciano , Anciano de 80 o más Años , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , Estudios Prospectivos , Dosificación Radioterapéutica , Neoplasias del Recto/patología , Neoplasias del Colon Sigmoide/patología , Tasa de SupervivenciaRESUMEN
Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3(-) T cells. These sequences are, however, demethylated in Foxp3(+) Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect and shown that transcription factors AP-1, Stat5, and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-ß signaling through Smad3-mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during induced Treg induction, whereas AP-1 and Creb maintain Enhancer 2 activity.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Acetilación , Animales , Secuencia de Bases , Sitios de Unión , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Histonas/metabolismo , Ratones , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Linfocitos T Reguladores/metabolismoRESUMEN
Glucocorticoid-induced TNFR (Gitr) and Ox40, two members of the TNFR superfamily, play important roles in regulating activities of effector and regulatory T cells (Treg). Their gene expression is induced by T cell activation and further upregulated in Foxp3+ Treg. Although the role of Foxp3 as a transcriptional repressor in Treg is well established, the mechanisms underlying Foxp3-mediated transcriptional upregulation remain poorly understood. This transcription factor seems to upregulate expression not only of Gitr and Ox40, but also other genes, including Ctla4, Il35, Cd25, all critical to Treg function. To investigate how Foxp3 achieves such upregulation, we analyzed its activity on Gitr and Ox40 genes located within a 15.1-kb region. We identified an enhancer located downstream of the Gitr gene, and both Gitr and Ox40 promoter activities were shown to be upregulated by the NF-κB-mediated enhancer activity. We also show, using the Gitr promoter, that the enhancer activity was further upregulated in conjunction with Foxp3. Foxp3 appears to stabilize NF-κB p50 binding by anchoring it to the enhancer, thereby enabling local accumulation of transcriptional complexes containing other members of the NF-κB and IκB families. These findings may explain how Foxp3 can activate expression of certain genes while suppressing others.
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Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Sitios Genéticos , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , FN-kappa B/metabolismo , Animales , Sitios de Unión , Complejo CD3/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Unión Proteica , Receptores OX40/genética , Elementos de Respuesta , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Activación TranscripcionalRESUMEN
The structure of ß-lactoglobulin (ß-LG) is well characterized, but the exact location of binding sites for retinol and (-)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic ß-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and ß-LG A with the most binding sites for EGCG, which include Tyr(20), Phe(151) and His(59). Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of ß-LG as nanotransporter.
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Catequina/análogos & derivados , Lactoglobulinas/química , Vitamina A/química , Animales , Sitios de Unión , Catequina/química , Bovinos , Dicroismo Circular , Cinética , Espectroscopía de Resonancia Magnética , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de FluorescenciaRESUMEN
INTRODUCTION: The different mechanisms: A. of the interaction between the molecular mother substance and the solvent water or ethanol B. of the storage of molecule-specific information in the solvent. C. the physiological basis of the sensitivity of the living organism towards an ultra high dilution (UHD). D. the mechanism of the interaction of the test dilution with the organism are largely unknown. Several ideas have been postulated, and experiments to test them carried out in physics and in biology. METHOD: The authors revisited a 1994 contribution on 'preliminary elements of a theory on UHDs' and updated it with regard to more recent literature and research findings. RESULTS: Although the experimental basis can still be questioned in most cases, remarkable fundamental observations have been made to explain the effects of UHDs. For some topics in question, it appears that information specific properties of the diluted substance to be transferred is stored by means electromagnetic fields. The interaction between the UHD and the organism seems to be electromagnetic in nature. The transmission of information from (bio-)molecules to the UHD is of special interest. Again, electromagnetic actions and vector potential fields appear to be implicated. CONCLUSION: The mechanisms of information storage and transfer in UHDs are far from fully understood, but progress has been made at experimental and theoretical levels.
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Homeopatía , Técnicas de Dilución del Indicador , Humanos , AguaRESUMEN
BACKGROUND: Since 1926, an influence of a dilution of silver nitrate (24x) on the growth of coleoptiles of wheat seedlings was described. The aim of the study discussed here is the critical proof of the reliability of a test system which has been quoted as a basic model for the research on homoeopathy for decades. METHODS: Grains of winter wheat (Triticum aestivum) were observed under the influence of extremely diluted silver nitrate (10(-23)) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy ('24x'). Analogously prepared water and/or inert water was used for control. Thirty experiments including 5000+5000 grains were performed by 5 researchers. RESULTS: Stalk lengths clearly indicate that development is enhanced by the probe silver nitrate 24x as compared to control. When the experiments 1989-1995 were pooled, means and SD for silver nitrate 24x-groups were 42.3±26.9 mm and for water control groups 34.7±22.2 mm. Verum stalk length was 21.9% bigger than control (100%) (p<0.01; d=0.31, i.e. small). For the experiments 1998-2014, means and SD were 73.7±21.7 mm and 60.5±20.9 mm. Verum stalk length was 21.7% bigger than control (100%) (p<0.01; d=0.62, i.e. medium). From the results one may hypothesize that the result is more marked in experiments showing an average mean of stalk length between ca. 50 and 90 mm in contrast to smaller or bigger mean lengths. CONCLUSION: The previous findings were confirmed by the results.
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Homeopatía/métodos , Reproducibilidad de los Resultados , Nitrato de Plata/administración & dosificación , Triticum/crecimiento & desarrollo , Humanos , Plantones/crecimiento & desarrolloRESUMEN
BACKGROUND: Following studies (a) on wheat seedlings and ultra high diluted silver nitrate, and (b) on amphibians and an ultra high diluted hormone, (c) a bio-assay on wheat and extremely diluted gibberellic acid was standardized. This assay was intended to combine the easy-to-handle aspect of (a) and biologically interesting aspects of (b). The purpose of the data analysis presented here was to investigate the influence of an extreme dilution of gibberellic acid on wheat stalk length and to determine the influence of external factors on the experimental outcome. METHODS: Grains of winter wheat (Triticum aestivum, Capo variety) were observed under the influence of extremely diluted gibberellic acid (10(-30)) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy ('G30x'). Analogously prepared water was used for control ('W30x'). 16 experiments including 8000+8000 grains were performed by 9 researchers. RESULTS: Experiments that were performed between January and April showed inconsistent results, whereas most of the experiments performed between September and December showed shorter stalks in the G30x group. This was confirmed by correlation analysis (p<0.01). Thus winter/spring experiments and autumn experiments were analysed separately. When all 10 autumn experiments were pooled, mean stalk lengths (mm) were 48.3±21.4 for the verum group and 52.1±20.4 for control (mean±SD) at grain level (N=5000 per group) and ±5.3 and ±5.1 respectively at dish level. In other words, verum stalk length (92.67%) was 7.33% smaller than control stalk length (100%). The effect size is small when calculation is done on the basis of grains (d=0.18) but, due to the smaller SD at dish level, medium when done on the basis of dishes (d=0.73). The inhibiting effect was observed by 6 of the 6 researchers who performed the autumn experiments. CONCLUSION: The model may be useful for further research as there exists a theoretical justification due to previous studies with wheat and extremely diluted silver nitrate, as well as to previous studies with amphibians and diluted hormones, and its methods are well standardized. Data confirm the hypothesis that information can be stored in the test liquid, even at a dilution of the original substance beyond Avogadro's value; and that the wheat bio-assay is sensitive to such information.
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Giberelinas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Plantones/efectos de los fármacos , Triticum/crecimiento & desarrollo , Homeopatía/métodos , Humanos , Plantones/crecimiento & desarrolloRESUMEN
INTRODUCTION: This paper focuses exclusively on experimental models with ultra high dilutions (i.e. beyond 10(-23)) that have been submitted to replication scrutiny. It updates previous surveys, considers suggestions made by the research community and compares the state of replication in 1994 with that in 2015. METHODS: Following literature research, biochemical, immunological, botanical, cell biological and zoological studies on ultra high dilutions (potencies) were included. Reports were grouped into initial studies, laboratory-internal, multicentre and external replications. Repetition could yield either comparable, or zero, or opposite results. The null-hypothesis was that test and control groups would not be distinguishable (zero effect). RESULTS: A total of 126 studies were found. From these, 28 were initial studies. When all 98 replicative studies were considered, 70.4% (i.e. 69) reported a result comparable to that of the initial study, 20.4% (20) zero effect and 9.2% (9) an opposite result. Both for the studies until 1994 and the studies 1995-2015 the null-hypothesis (dominance of zero results) should be rejected. Furthermore, the odds of finding a comparable result are generally higher than of finding an opposite result. Although this is true for all three types of replication studies, the fraction of comparable studies diminishes from laboratory-internal (total 82.9%) to multicentre (total 75%) to external (total 48.3%), while the fraction of opposite results was 4.9%, 10.7% and 13.8%. Furthermore, it became obvious that the probability of an external replication producing comparable results is bigger for models that had already been further scrutinized by the initial researchers. CONCLUSIONS: We found 28 experimental models which underwent replication. In total, 24 models were replicated with comparable results, 12 models with zero effect, and 6 models with opposite results. Five models were externally reproduced with comparable results. We encourage further replications of studies in order to learn more about the model systems used.
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Bibliometría , Homeopatía/métodos , Homeopatía/normas , Técnicas de Dilución del Indicador/normas , Modelos Teóricos , HumanosRESUMEN
BACKGROUND: A model of thyroxine and metamorphosis of highland amphibians is frequently mentioned as an example of experiments on extremely diluted substances in discussions around 'homeopathy'. METHODS: The model was scrutinized by reanalysing the results of the initial researcher A and a second researcher B as well as of 5 external researchers C between 1990 and 2013. Rana temporaria larvae were taken from an alpine highland biotope. The test solution was thyroxine 10(-30) (T30x), tetra-iodo-thyronine sodium pentahydrate diluted with pure water in 26 steps of 1:10, being agitated after each step. Analogously prepared water (W30x) was used for control. Tadpoles were observed from the 2-legged to the 4-legged stage. Experiments were performed in different years, at different times of season, and their duration could vary. Frequencies of 4-legged animals, effect sizes and areas under the curves (AUCs) were calculated and regression analyses were performed to investigate possible correlations between year, season, duration etc. Experiments were in line with animal protection guidelines. RESULTS: The total set of data A + B + C as well as subsets A (initial researcher, N=286+293), B (second centre, 965 + 965) and C (5 external researchers, 690 + 690) showed an effect of extremely diluted agitated thyroxine reverse to that known of molecular thyroxin, i.e. test values were below control by 11.4% for A, 9.5% for B and 7.0% for C (p<0.001 for each of the subsets). The effect size (Cohen's d) was >0.8 (large) for both A and B and 0.74 (medium) for C. CONCLUSION: Although a perfect reproducibility was not obtained, this paradoxical phenomenon was generally consistent in different observations. Correlations were found between details of laboratory handling, as well as environment temperature, and the size of the results.
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Anfibios/crecimiento & desarrollo , Homeopatía/métodos , Metamorfosis Biológica/efectos de los fármacos , Tiroxina/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Larva/crecimiento & desarrollo , Reproducibilidad de los Resultados , Tiroxina/administración & dosificaciónRESUMEN
In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.
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Células Dendríticas/ultraestructura , Exosomas/ultraestructura , Proteínas PrPC/análisis , Scrapie/patología , Animales , Separación Celular , Células Dendríticas/metabolismo , Exosomas/metabolismo , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Proteínas PrPC/metabolismo , Proteínas PrPC/ultraestructura , Scrapie/metabolismo , Bazo/metabolismo , Bazo/patologíaRESUMEN
BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.
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Virus de la Fiebre Porcina Clásica/genética , Virus ARN/genética , ARN/genética , Recombinación Genética , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , Virus de la Fiebre Porcina Clásica/crecimiento & desarrollo , Clonación Molecular , ADN Complementario/genética , Mutagénesis , Sistemas de Lectura Abierta , Virus ARN/crecimiento & desarrollo , PorcinosRESUMEN
Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.
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Virus de la Fiebre Porcina Clásica/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Virus de la Fiebre Porcina Clásica/genética , Escherichia coli/genética , Genes Reporteros , Luciferasas/análisis , Luciferasas/genética , Biosíntesis de Proteínas , ARN Viral/genética , ARN Viral/metabolismo , Recombinación Genética , Replicón , Ovinos , Coloración y Etiquetado/métodos , Transcripción GenéticaRESUMEN
BACKGROUND AND PURPOSE: Enterococci are emerging causes of severe infections, including wound and bone infections in orthopedic patients. The main purpose of this study was to determine whether there was a correlation between the incidence of enterococci in tissue samples (biopsies) from orthopedic patients and consumption of cefuroxime in the orthopedic department. METHODS AND RESULTS: Data were obtained from the department of clinical microbiology and the hospital pharmacy. The consumption of cefuroxime successively increased from 40 defined daily doses (DDD)/10(3) bed days in 2002 to 212 DDD/10(3) bed days in 2009. The incidence of patients with enterococci in tissue samples increased steadily from 1.03/10(3) bed days in 2002 to 5.90/10(3) bed days in 2009. Regression analysis revealed a significant correlation between the consumption of cefuroxime and the incidence of enterococci. INTERPRETATION: Continuous surveillance of species distribution, resistance rates, and antibiotic consumption is of utmost importance for optimal antibiotic strategy in orthopedic patients.
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Antibacterianos/administración & dosificación , Cefuroxima/administración & dosificación , Enterococcus/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Procedimientos Ortopédicos , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Dinamarca/epidemiología , Utilización de Medicamentos/estadística & datos numéricos , Utilización de Medicamentos/tendencias , Enterococcus/clasificación , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Incidencia , Estudios RetrospectivosRESUMEN
PURPOSE: In childhood and adolescence, cross-sectional imaging, most commonly computed tomography (CT), is often performed for advanced diagnosis of joint injuries of the distal lower leg and upper ankle. Due to radiation exposure, the need for CT remains controversial, as these injuries follow stereotypies and usually have a similar course. Alternatively, the performance of magnetic resonance imaging (MRI) is also discussed. Since radiation sensitivity at this young age is much higher than in adults, an effort must be to minimize radiation exposure according to as low as reasonably achievable (ALARA) principles. The aim of this survey is to evaluate the current procedure in Germany in the diagnosis of pediatric injuries of the distal lower leg and upper ankle. METHODS: For data collection, a survey entitled "CT in fractures of the ankle joint in childhood and adolescence: subject of the survey are injuries between 8 and 15 years of age" of the Section of Pediatric Traumatology in the German Association of Trauma Surgery was sent to all members via the distribution list of the German Society of Orthopedics and Traumatology and the distribution list of the German Society of Pediatric Surgery in a period from September 20, 2022-December 21, 2022. The survey included a total of 21 questions. Target groups were trauma and pediatric surgeons and orthopedic surgeons working in the hospital and in practice. RESULTS: A total of 525 participants took part in the survey: ultrasound diagnostics are used by almost 25% and the Ottawa Ankle Rules by over 50% always or in most cases. A conventional x-ray is always or most often used by over 90%. CT imaging is rarely used by 88.57%, mainly for surgical planning or analysis of fracture progression. 69.9% report that their radiology department uses a pediatric protocol for CT exams; 25.71% do not know if this is the case. MRI imaging is also used infrequently by 89.33%, mostly to identify associated injuries. Overall, CT imaging is chosen by 55.62% and MRI imaging by 35.24% as the sectional imaging modality for suspected fractures; 95.05% consider sectional imaging useful for a triplane fracture, 59.24% for a two-plane fracture, 41.71% for a Salter-Harris type III/IV injury, and 8% for a Salter-Harris type I/II injury. CONCLUSION: The survey showed that the conventional X-ray is still the gold standard. Interestingly, more than half of the respondents regularly use the Ottawa Ankle Rules, and diagnostics using ultrasound are also used by almost a quarter. Awareness of radiation protection in children exists, although a quarter of all participants do not know the extent to which their radiology department has a specific pediatric protocol for CT imaging. Cross-sectional imaging is performed on a regular basis. Regarding the actual extent of imaging, there is a clear divergence between theory and practice.