Host Cell Targets for Unconventional Antivirals against RNA Viruses.

The recent COVID-19 crisis has highlighted the importance of RNA-based viruses. The most prominent members of this group are SARS-CoV-2 (coronavirus), HIV (human immunodeficiency virus), EBOV (Ebola virus), DENV (dengue virus), HCV (hepatitis C virus), ZIKV (Zika virus), CHIKV (chikungunya virus), and influenza A virus. With the exception of retroviruses which produce reverse transcriptase, the majority of RNA viruses encode RNA-dependent RNA polymerases which do not include molecular proofreading tools, underlying the high mutation capacity of these viruses as they multiply in the host cells. Together with their ability to manipulate the immune system of the host in different ways, their high mutation frequency poses a challenge to develop effective and durable vaccination and/or treatments. Consequently, the use of antiviral targeting agents, while an important part of the therapeutic strategy against infection, may lead to the selection of drug-resistant variants. The crucial role of the host cell replicative and processing machinery is essential for the replicative cycle of the viruses and has driven attention to the potential use of drugs directed to the host machinery as therapeutic alternatives to treat viral infections. In this review, we discuss small molecules with antiviral effects that target cellular factors in different steps of the infectious cycle of many RNA viruses. We emphasize the repurposing of FDA-approved drugs with broad-spectrum antiviral activity. Finally, we postulate that the ferruginol analog (18-(phthalimide-2-yl) ferruginol) is a potential host-targeted antiviral.

Efficacy and safety of FOLFIRI/aflibercept (FA) in an elderly population with metastatic colorectal cancer (mCRC) after failure of an oxaliplatin-based regimen.

BACKGROUND: The VELOUR study showed the benefit of FOLFIRI-Aflibercept (FA) versus FOLFIRI in patients with metastatic colorectal cancer (mCRC) in second-line treatment. However, only 36% of the included patients were ≥65 years. Thus, we seek to evaluate the efficacy and safety of FA in the elderly population in the context of routine practice. MATERIALS AND METHODS: We conducted an observational, retrospective, multicenter, observational study of patients ≥70 years with mCRC treated with FA after progression to oxaliplatin chemotherapy in routine clinical practice in 9 hospitals of the GITuD group. RESULTS: Of 388 patients treated with FA between June 2013 and November 2018, 75 patients ≥70 years were included. The median number of cycles was 10 and the objective response (ORR) and disease control rates (DCR) were 33.8% and 72.0%, respectively. With a median follow-up of 27.1 months, median Progression-free survival (PFS) was 6.6 months and median Overall Survival (OS) was 15.1 months. One third fewer metastasectomies were performed in the ≥75 years' subgroup (24 vs. 52%, p = 0.024) and more initial FOLFIRI dose reductions (68 vs. 36%, p = 0.014). ORR (23.8% vs. 38.3%), DCR (42.8% vs. 85.1%), and PFS (4 vs. 7.8 months; p = 0.017) were significantly less, without difference in OS (9.9 vs. 17.1 months; p = 0.129). The presence of prior hypertension (HT) (PFS 7.9 vs. 5.7 months, p = 0.049) and HT ≥ grade 3 during treatment (PFS 7.6 vs. 6.6 months, p = 0.024) were associated with longer PFS. The most frequent grade 3/4 adverse events were: asthenia (21.3%), neutropenia (14.7%), and diarrhea (14.7%). 57.3% required FOLFIRI dose reduction; 34.7% of aflibercept, including discontinuation (5.3% and 18.7%, respectively). CONCLUSIONS: FA combination is effective in patients ≥70 years. The occurrence of HT is predictive of efficacy. Close monitoring of toxicity and initial dose adjustment is recommended.

Depresión y ansiedad y su relación con el perfil antropométrico de los pacientes en rehabilitación cardíaca fases I y II / Depression and anxiety and their relationship with the anthropometric profile of patients in Cardiac Rehabilitation Phases I and II

RESUMEN Introducción: La depresión es una alteración del estado mental que afecta a muchas personas alrededor del mundo y que, junto con la ansiedad, constituye un problema a nivel mundial que puede afectar a los pacientes en el período posquirúrgico cardiovascular. Objetivo: Determinar los niveles de depresión y ansiedad, y su relación con el sobrepeso y la obesidad, en pacientes que asisten a rehabilitación cardíaca fases I y II. Método: Se hizo la selección de 50 participantes de rehabilitación cardíaca (25 de fase I y 25 en fase II). Se utilizó la Hospital Anxiety and Depression Scale (HADS) para la detección de trastornos de ansiedad y depresión. Además, se valoró la antropometría de los participantes y se realizaron pruebas de normalidad de Kolmogorov-Smirnov y Shapiro-Wilk; como también, la media, desviación estándar y el coeficiente de correlación de Pearson con un grado significativo de p<0,05. Resultados: Los 50 participantes (66% hombres) tenían una edad promedio de 63,86±10,99, con diagnósticos posoperatorios de revascularización miocárdica (44%), angioplastia coronaria (40%), enfermedad aterosclerótica (4%), reemplazo de válvula aórtica (4%), cierre de comunicación interauricular (4%), marcapasos implantado (2%) y desacondicionamiento físico (2%). Se encontró una depresión de 36% y ansiedad de 30%. Conclusiones: Existe una alta prevalencia de depresión y ansiedad en los programas de rehabilitación cardíaca, su frecuencia es mayor en la fase I en comparación con la II. Además, se encontró que existe una correlación moderada leve entre la ansiedad y el normopeso y la obesidad, al igual que entre la depresión frente al sobrepeso.

ABSTRACT Introduction: Depression is a mental state disorder that affects a good number of people around the world and that, along with anxiety, is a wide-reaching problem that can strike patients after undergoing heart surgery. Objective: To determine the levels of depression and anxiety, and their relationship with overweight and obesity, in patients attending cardiac rehabilitation phases I and II. Method: Fifty patients receiving cardiac rehabilitation (25 in phase I and 25 in phase II) were selected. The Hospital Anxiety and Depression Scale (HADS) was used to screen anxiety and depression disorders. In addition, the anthropometry of the participants was examined and Kolmogorov-Smirnov and Shapiro-Wilk normality tests were performed. Mean, standard deviation and Pearson correlation coefficient with a significant degree of p<0.050 were also applied. Results: The 50 participants (66% men) had an average age of 63.86±10.99, with postoperative diagnosis of coronary-artery bypass grafting (44%), coronary angioplasty (40%), atherosclerotic disease (4%), aortic valve replacement (4%), atrial septal defect closure (4%), implanted pacemaker (2%) and physical deconditioning (2%). Depression was found at 36% and anxiety at 30%. Conclusions: There is a high prevalence of depression and anxiety in cardiac rehabilitation programs; its frequency is higher in phase I compared to phase II. Moreover, we found that there is a slight-mild correlation between anxiety versus normal weight and obesity, as well as depression versus overweight.

Anti-herpetic and anti-dengue activity of abietane ferruginol analogues synthesized from (+)-dehydroabietylamine.

The abietane-type diterpenoid (+)-ferruginol (1), a bioactive compound isolated from several plants, has attracted much attention as consequence of its pharmacological properties, which includes antibacterial, antifungal, antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and antitumor actions. In this study, we report on the antiviral evaluation of ferruginol (1) and several analogues synthesized from commercial (+)-dehydroabietylamine. Thus, the activity against Human Herpesvirus type 1, Human Herpesvirus type 2 and Dengue Virus type 2, was studied. Two ferruginol analogues showed high antiviral selectivity index and reduced viral plaque-size in post-infection stages against both Herpes and Dengue viruses. A promising lead, compound 8, was ten-fold more potent (EC50 = 1.4 µM) than the control ribavirin against Dengue Virus type 2. Our findings suggest that the 12-hydroxyabieta-8,11,13-triene skeleton, which is characteristic of the diterpenoid ferruginol (1), is an interesting molecular scaffold for development of novel antivirals. In addition, the cytotoxic and antifungal activities of the synthesized ferruginol analogues have also been investigated. (©)20155 Elsevier Science. All rights reserved.

Performance characteristics of loci method for measuring cardiac troponin I on the dimension EXL.

OBJECTIVE: To define the performance characteristics of the LOCI® method for cardiac troponin I on the Dimension EXL system. DESIGNS AND METHODS: Three different levels of commercial control (mean concentrations 0.426, 1.42, and 18.64 µg/L) were used for the imprecision study, quantifying separately within-run and between-run over 20 days. The limit of blank (LoB) and limit of detection (LoD) were assessed with 20 replicates of a sample without troponin I. Linearity was assessed by regression analysis. In addition, we studied inaccuracy, carry-over and limit of quantitation and conducted a method comparison with the Stratus CS (n=69). The reference interval was determined in 146 healthy blood donors using non-parametric method. RESULTS: The within-run imprecision (coefficient of variation [CV], %) obtained at each level was 2.4, 1.4% and 2.2%, while the between-run imprecision (CV,%) was 3.3%, 2.9% and 2.5%. Total imprecision was 4.06%, 3.3% and 3.4% for each control level. The limit of quantitation which corresponds to the troponin I concentration at which CV=10% was 0.05 µg/L. Method comparison with the Stratus CS assay produced the equation: Dimension EXL=-0.002698+1.0233⁎(Stratus CS) with a confidence interval from -0.01562 to 0.00626 for the intercept and (0.979 to 1.0875) for the slope. The 99th percentile obtained for the reference population was 0.047 µg/L. CONCLUSIONS: The LOCI method for cardiac troponin I on the Dimension EXL meets all guidelines recommended criteria referring to limit of quantitation, imprecision and shows excellent transferability with the Stratus CS method.

Computational Identification of Dengue Virus MicroRNA-Like Structures and their Cellular Targets.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate transcriptional and posttranscriptional gene regulation of the cell. Experimental evidence shows that miRNAs have a direct role in different cellular processes, such as immune function, apoptosis, and tumorigenesis. In a viral infection context, miRNAs have been connected with the interplay between host and pathogen, occupying a major role in pathogenesis. While numerous viral miRNAs from DNA viruses have been identified, characterization of functional RNA virus-encoded miRNAs and their potential targets is still ongoing. Here, we used an in silico approach to analyze dengue Virus genome sequences. Pre-miRNAs were extracted through VMir software, and the identification of putative pre-miRNAs and mature miRNAs was accessed using Support Vector Machine web tools. The targets were scanned using miRanda software and functionally annotated using ClueGo. Via computational tools, eight putative miRNAs were found to hybridize with numerous targets of morphogenesis, differentiation, migration, and growth pathways that may play a major role in the interaction of the virus and its host. Future approaches will focus on experimental validation of their presence and target messenger RNA genes to further elucidate their biological functions in human and mosquito cells.

Lovastatin delays infection and increases survival rates in AG129 mice infected with dengue virus serotype 2.

BACKGROUND: It has been reported that treatment of DENV-infected cultures with Lovastatin (LOV), can affect viral assembly. The objective of this study was to evaluate the effect of LOV on the survival rate and viremia levels of DENV-2-infected AG129 mice. METHODOLOGY/PRINCIPAL FINDINGS: Mice were inoculated with 1 × 10(6) plaque-forming units (PFU/ml) of DENV-2 and treated with LOV (200 mg/kg/day). Pre-treatment with one or three doses of LOV increased the survival rate compared to untreated mice (7.3 and 7.1 days, respectively, compared to 4.8 days). Viremia levels also decreased by 21.8% compared to untreated mice, but only in the group administered three doses prior to inoculation. When LOV was administered after viral inoculation, the survival rate increased (7.3 days in the group treated at 24 hpi, 6.8 days in the group treated at 48 hpi and 6.5 days in the group treated with two doses) compared to the untreated group (4.8 days). Interestingly, the serum viral titer increased by 24.6% in mice treated at 48 hpi with a single dose of LOV and by 21.7% in mice treated with two doses (at 24 and 48 hpi) of LOV compared to untreated mice. Finally histopathological changes in the liver and spleen in infected and untreated mice included massive extramedullary erythropoiesis foci and inflammatory filtration, and these characteristics were decreased or absent in LOV-treated mice. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the effect of LOV on viremia depends on the timing of treatment and on the number of doses administered. We observed a significant increase in the survival rate in both schemes due to a delay in the progression of the disease. However, the results obtained in the post-treatment scheme must be handled carefully because this treatment scheme increases viremia and we do not know how this increase could affect disease progression in humans.

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome.

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs.

Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs.

Utilidad del EuroSCORE en la predicción de mortalidad intrahospitalaria en una institución de enfermedades cardiovasculares de Colombia / Usefulness of EuroSCORE in predicting hospital mortality in an institution of cardiovascular diseases in Colombia

Introducción: en ausencia de datos nacionales sobre el tema, los autores se propusieron evaluar la utilidad del European System for Cardiac Operative Risk Evaluation (EuroSCORE) en la predicción de mortalidad intrahospitalaria en una institución cardiovascular de Colombia. Métodos: desde agosto de 2008 a diciembre de 2010, se siguió una cohorte de 750 pacientes adultos sometidos a procedimientos como: revascularización miocárdica, cambios valvular aórtico o mitral u otras cirugías cardiacas. Para todos ellos se calculó el EuroSCORE logístico y se estimó su relación con la mortalidad intrahospitalaria. Resultados: la mortalidad observada fue significativamente inferior a la esperada según el EuroSCORE (5,9% vs. 8,1%; RR = 0,73; IC95%: 0,51-0,94; p=0,03). El EuroSCORE mostró un área bajo la curva ROC de 86,1% (IC95%: 80,4%-90,7%) para la predicción de este evento. Se observó un patrón de incremento de la mortalidad por un factor de 1,07 (IC95%: 1,05-1,08; p<0,001) por cada cambio de un punto en el EuroSCORE. Al agrupar los pacientes sometidos a revascularización y cambios valvulares, el área bajo la curva ROC del EuroSCORE alcanzó el 90,3% (IC95%: 80,3%-100%), superior a lo observado en otros procedimientos (80,9%; IC95%: 72,7%-89%). Sin embargo, esta diferencia no alcanzó significancia estadística (p=0,15). Conclusiones: EuroSCORE fue útil para predecir mortalidad; sin embargo, los valores observados estuvieron por debajo de los esperados y su utilidad pareció variar de acuerdo con la intervención. Por tanto, se justifica realizar estudios de mayor magnitud para validar esta escala en diferentes procedimientos o proponer una escala basada en datos locales.

Introducción: en ausencia de datos nacionales sobre el tema, los autores se propusieron evaluar la utilidad del European System for Cardiac Operative Risk Evaluation (EuroSCORE) en la predicción de mortalidad intrahospitalaria en una institución cardiovascular de Colombia. Métodos: desde agosto de 2008 a diciembre de 2010, se siguió una cohorte de 750 pacientes adultos sometidos a procedimientos como: revascularización miocárdica, cambios valvular aórtico o mitral u otras cirugías cardiacas. Para todos ellos se calculó el EuroSCORE logístico y se estimó su relación con la mortalidad intrahospitalaria. Resultados: la mortalidad observada fue significativamente inferior a la esperada según el EuroSCORE (5,9% vs. 8,1%; RR = 0,73; IC95%: 0,51-0,94; p=0,03). El EuroSCORE mostró un área bajo la curva ROC de 86,1% (IC95%: 80,4%-90,7%) para la predicción de este evento. Se observó un patrón de incremento de la mortalidad por un factor de 1,07 (IC95%: 1,05-1,08; p<0,001) por cada cambio de un punto en el EuroSCORE. Al agrupar los pacientes sometidos a revascularización y cambios valvulares, el área bajo la curva ROC del EuroSCORE alcanzó el 90,3% (IC95%: 80,3%-100%), superior a lo observado en otros procedimientos (80,9%; IC95%: 72,7%-89%). Sin embargo, esta diferencia no alcanzó significancia estadística (p=0,15). Conclusiones: EuroSCORE fue útil para predecir mortalidad; sin embargo, los valores observados estuvieron por debajo de los esperados y su utilidad pareció variar de acuerdo con la intervención. Por tanto, se justifica realizar estudios de mayor magnitud para validar esta escala en diferentes procedimientos o proponer una escala basada en datos locales.

Implementación de un control interno en la detección molecular de las principales especies de micoplasmas contaminantes de cultivos celulares / Implementation of an internal control in molecular detection of the main mycoplasma species contaminating cell cultures

Objetivo: Construir e implementar un control interno para la detección molecular de micoplasmas en cultivos celulares. Materiales y métodos: Se alinearon secuencias del RNA ribosomal 16S de las principales especies de micoplasmas reportadas como contaminantes de cultivos, y diseñaron oligonucleótidos para la detección de las mismas. Para la construcción del control interno se amplificó una secuencia espadadora, además de las secuencias de hibridación de los oligonucleótidos usados para detección. La amplificación desde dicho control generó un fragmento con tamaño diferente al obtenido desde una muestra positiva. Posteriormente, para la evaluación del efecto del control interno sobre la amplificación de una muestra positiva se realizó la prueba sobre diluciones seriadas de la muestra, en presencia y ausencia del control interno. Finalmente, se ensayó el protocolo utilizando sobrenadantes de cultivo provenientes de diferentes laboratorios. Resultados: Se observó alta homogeneidad al comparar los géneros Mycoplasma y Acholeplasma; no obstante se realizó el diseño de oligonucleótidos degenerados para aumentar teóricamente la eficiencia en la detección de todas las especies. Se demostró que la aplicación del control interno no afecta la sensibilidad de detección de la prueba. Los resultados obtenidos con muestras problema resaltan la importancia del control interno al realizar la prueba desde sobrenadantes de cultivo. Conclusiones: El uso del control interno evita la obtención de resultados falsos negativos, generados por presencia de inhibidores en la muestra. La implementación de la prueba beneficiará los laboratorios en cuyas investigaciones utilicen cultivos celulares y permitirá ejercer un control de calidad, lo cual hará más confiables los resultados/productos obtenidos.

Objective: to construct and implement an internal control for molecular detection of mycoplasma in cell cultures. Materials and Methods: Sequences of 16S ribosomal RNA of the major species of mycoplasma reported as contaminants in cell cultures were aligned, and oligonucleotides for detection were designed. For the construction of the internal control, a spacer sequence as well as sequences for hybridization of the oligonucleotides used for detection, were amplified. Amplification from the internal control and positive samples generated fragments with different sizes. Subsequently, to evaluate the effect of the internal control on the amplification of a positive sample, the assay was performed on serial dilutions of the sample in the presence and absence of the internal control. Finally, the protocol was tested using culture supernatants from different laboratories. Results: High homogeneity was observed when Mycoplasma and Acholeplasma genera were compared; however, degenerated oligonucleotides were designed to increase the efficiency of detection in all the analyzed species. It was shown that implementation of the internal control does not affect the sensitivity of detection. The results obtained with cell cultures samples highlighted the importance of the internal control. Conclusions: The use of an internal control facilitates the detection of false negative results generated by the presence of inhibitors in the sample. Implementation of the test as a quality control will benefit laboratories using cell cultures, making the results and / or products obtained more reliable.

Detección molecular y tipificación del virus dengue por RT-PCR y PCR anidada usando oligonucleótidos mejorados / Molecular detection and typing of dengue virus by RT-PCR and nested PCR using degenerated oligonucleotides

Objetivos: Modificar los oligonucleótidos comúnmente utilizados para la detección y tipificación del virus dengue y evaluar su desempeño sobre ARNs provenientes de muestras clínicas. Materiales y métodos: A partir del alineamiento de secuencias disponibles en el GenBank para la región C-prM/M se determinó la variabilidad genética dentro de cada serotipo del virus dengue, lo cual permitió realizar modificaciones a los oligonucleótidos convencionalmente usados en la tipificación. Tales modificaciones incluyeron la adición y eliminación de nucleótidos en el extremo 3', teniendo en cuenta la posición del codón, así como la inclusión de sitios degenerados en posiciones altamente variables. Los oligonucleótidos se evaluaron a diferentes concentraciones sobre ARNs extraídos a partir de cultivos celulares infectados y posteriormente de sueros de pacientes con diagnóstico probable de dengue. Resultados: Los oligonucleótidos amplificaron específicamente cada serotipo del virus dengue, tanto desde sobrenadantes de cultivo como desde muestras clínicas en prueba piloto. Conclusiones: El rediseño de los oligonucleótidos consideró la variabilidad genética acumulada en más de 15 años, mostrándose experimentalmente su valor y utilidad en la detección y tipificación del virus dengue.

Objective: To modify the commonly used primers for detecting and typing of dengue viruses and evaluate their performance on RNAs derived from clinical samples. Materials and methods: To determine the genetic variability within each dengue virus serotype, sequences of C-prM/M region available on GenBank were aligned allowing modifications to the primers conventionally used for virus typing. Modifications include nucleotide insertion and deletion in the 3' end taking into account the codon position, and also the inclusion of degenerated sites in highly variable positions. Primers were evaluated at different concentrations on extracted RNAs from infected cell cultures and subsequently from sera of probable dengue diagnosed patients. Results: All primers specifically amplified each dengue virus serotype from cell culture supernatants and clinical samples in a pilot test. Conclusions: The re-designed primers took into account the accumulated variability during more than 15 years, experimentally showing their value and utility for detecting and typing of dengue viruses.

Phylogenetic reconstruction of dengue virus type 2 in Colombia.

BACKGROUND: Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and sub-tropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2) has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E) gene from 48 Colombian isolates. RESULTS: As expected, the oldest isolates belonged to the American genotype (subtype V), but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb) as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. CONCLUSION: After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated.

Risk of fracture in celiac disease: gender, dietary compliance, or both?

AIM: To determine the incidence of peripheral fractures in patients with celiac disease (CD) and the effect of treatment on fracture risk. METHODS: We compared the incidence and risk of peripheral fractures before and after diagnosis between a cohort of 265 patients who had been diagnosed with CD at least 5 years before study entry and a cohort of 530 age- and sex-matched controls who had been diagnosed with functional gastrointestinal disorders. Data were collected through in-person interviews with an investigator. The overall assessment window for patients was 9843 patient-years (2815 patient-years after diagnosis). RESULTS: Compared with the control group, the CD cohort showed significantly higher incidence rate and risk of first peripheral fracture before diagnosis [adjusted hazard ratio (HR): 1.78, 95% CI: 1.23-2.56, P < 0.002] and in men (HR: 2.67, 95% CI: 1.37-5.22, P < 0.004). Fracture risk was significantly associated with the classic CD presentation with gastrointestinal symptoms (P < 0.003). In the time period after diagnosis, the risk of fractures was comparable between the CD cohort and controls in both sexes (HR: 1.08, 95% CI: 0.55-2.10 for women; HR: 1.57, 95% CI: 0.57-4.26 for men). CONCLUSION: CD patients have higher prevalence of fractures in the peripheral skeleton before diagnosis. This is associated with male sex and classic clinical presentation. The fracture risk was reduced after the treatment.

Statins reduce dengue virus production via decreased virion assembly.

BACKGROUND: Most of the effects of statins can be explained by pleiotropic effects independent of their lowering of serum cholesterol; in some cases, these effects have been shown to be a result of the role of statins in the prenylation of cellular proteins, some of which are involved in the life cycle of animal viruses. This study evaluated the potential antiviral activity of lovastatin (LOV) against dengue virus (DENV) infection of epithelial and endothelial cells (VERO cells, epithelial cells derived from African green monkey kidney, and HMEC-1 cells, human dermal microvascular endothelial cells). METHODS: To evaluate its potential antiviral effects, LOV was used before, during and after inoculation of cell cultures with DENV. RESULTS: Before and after viral inoculation, LOV caused a reduction in virus yield (80% for HMECs and 25% for VERO cells). However, with LOV treatment after inoculation induced a marked increase (2- to 9-fold) in viral-positive RNA while the amount of viral protein increased only by 13-23%. A moderate reduction (1 log unit) in viral titer occurred concurrent with the increase in DENV genomic RNA and protein within the cells. CONCLUSIONS: According to our results, LOV appears to have a greater effect on viral assembly than on replication, resulting in the cellular presence of viral genomic RNA and proteins that fail to take the normal assembly pathway.

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia.

BACKGROUND: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70's when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. RESULTS: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major amino acid changes in the analyzed region were found. CONCLUSION: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.

Celiac disease serology in patients with different pretest probabilities: is biopsy avoidable?

AIM: To establish the diagnostic performance of several serological tests, individually and in combination, for diagnosing celiac disease (CD) in patients with different pretest probabilities, and to explore potential serological algorithms to reduce the necessity for biopsy. METHODS: We prospectively performed duodenal biopsy and serology in 679 adults who had either high risk (n = 161) or low risk (n = 518) for CD. Blood samples were tested using six assays (enzyme-linked immunosorbent assay) that detected antibodies to tissue transglutaminase (tTG) and deamidated gliadin peptide (DGP). RESULTS: CD prevalence was 39.1% in the high-risk population and 3.3% in the low-risk group. In high-risk patients, all individual assays had a high diagnostic efficacy [area under receiving operator characteristic curves (AU ROC): 0.968 to 0.999]. In contrast, assays had a lower diagnostic efficacy (AU ROC: 0.835 to 0.972) in the low-risk group. Using assay combinations, it would be possible to reach or rule out diagnosis of CD without biopsy in 92% of cases in both pretest populations. We observed that the new DGP/tTG Screen assay resulted in a surplus compared to more conventional assays in any clinical situation. CONCLUSION: The DGP/tTG Screen assay could be considered as the best initial test for CD. Combinations of two tests, including a DGP/tTG Screen, might be able to diagnose CD accurately in different clinical scenarios making biopsy avoidable in a high proportion of subjects.

ERK2, but not ERK1, mediates acquired and "de novo" resistance to imatinib mesylate: implication for CML therapy.

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.

A prospective evaluation of endoscopic markers for identifying celiac disease in patients with high and low probability of having the disease.

BACKGROUND/OBJECTIVES: the usefulness of duodenoscopic markers for predicting celiac disease (CD) has been questioned. We assessed the diagnostic efficacy of endoscopic markers of mucosal atrophy in individuals with different pretest probability of CD. METHODS: we prospectively performed endoscopic intestinal biopsies and CD-related serology tests in 661 individuals, including 143 consecutive patients attending a malabsorption clinic (high pretest probability) and 518 subjects randomly selected fom those undergoing routine endoscopy because of upper GI symptoms (low pretest probability). Duodenoscopic markers reported were: mosaic pattern, scalloped folds, and reduction in number or loss of Kerkring's folds. RESULTS: sixty-three (44.1%) and 18 (3.5%) patients were diagnosed with CD in the high and low risk groups, respectively Among high pretest subjects, the presence of any marker had very high sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy for the identification of CD (92.1%, 93.8%, 92.1%, 93.8% and 93.0%, respectively). The performance of these parameters for the presence of any marker in the low pretest population were 61.1%, 96.8%, 40.7%, 98.6% and 95.6%, respectively. Sensitivity (p < 0.004) and positive predictive value (p < 0.0001) of markers were significantly higher for the high risk patients. The identification of a reduction in number or loss of Kerkring'sfolds was not a reliable finding unless other signs were also present. CONCLUSIONS: we confirm that endoscopic markers are useful in predicting CD in different clinical scenarios. The high negative predictive value in the low probability group suggests that intestinal biopsy is not required if endoscopic markers are absent.