Comparative proteomic analysis of patients with acute myeloid leukemia before and after induction therapy.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Response to chemotherapy treatment in patients with AML is wide-ranging, and to date there are no adequate molecular biomarkers used to predict clinical outcome. OBJECTIVE: The aim of this study was to identify potential protein biomarkers which could help predict response to induction treatment in AML patients. METHODS: Peripheral blood samples were obtained from 15 AML patients both before and after treatment. A comparative proteomic analysis was performed using 2D gel electrophoresis followed by Mass Spectrometry. RESULTS: This comparative proteomic study, combined with a protein network analysis, revealed several proteins that could be considered potential biomarkers of poor prognosis in AML: GAPDH which favors increased glucose metabolism; eEF1A1 and Annexin A1 that promote proliferation and migration, cofilin 1 which plays a role in the activation of apoptosis; and GSTP1 which is involved in the processes of detoxification and chemoresistance. CONCLUSIONS: This study gives an insight into a panel of protein biomarkers with prognostic potential that should be further investigated.

Deciphering the role of Wnt signaling in acute myeloid leukemia prognosis: how alterations in DNA methylation come into play in patients' prognosis.

Acute myeloid leukemia (AML) is a malignant clonal disorder affecting myeloid differentiation through mechanisms that include epigenetic dysregulation. Abnormal changes in DNA methylation and gene expression profiles of pathways involved in hematopoietic development, such as Wnt/ß-catenin, contribute to the transformation, development, and maintenance of leukemic cells. This review summarizes the alterations of Wnt signaling-related genes at the epigenetic and transcriptional level and their implications for AML prognosis. Among the implications of epigenetic alterations in AML, methylation of Wnt antagonists is related to poor prognosis, whereas their upregulation has been associated with a better clinical outcome. Furthermore, Wnt target genes c-Myc and LEF-1 present distinct implications. LEF-1 expression positively influences the patient overall survival. c-Myc upregulation has been associated with treatment resistance in AML, although c-Myc expression is not exclusively dependent of Wnt signaling. Understanding the signaling abnormalities could help us to further understand leukemogenesis, improve the current risk stratification for AML patients, and even serve to propose novel therapeutic targets.

Bevacizumab as a treatment option in gastrointestinal bleeding associated to hereditary hemorrhagic telangiectasia. Case Report / Bevacizumab como opción terapéutica en sangrado gastrointestinal asociado a telangiectasia hemorrágica hereditaria. Reporte de caso

Abstract Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disease characterized by the presence of arteriovenous malformations in the nasal mucosa, the tips of fingers, and sometimes in the lungs, the gastrointestinal tract, the liver, the pancreas, the marrow and the brain. Its treatment is based on symptomatic control measures, but recently, the administration of anti-vascular endothelial growth factor (VEGF) molecules has been proposed as a treatment alternative, especially in patients with recurrent bleeding. Case presentation: The case of a 67-year-old man diagnosed with HHT and suffering from potentially life-threatening gastrointestinal GI bleeding is presented. The patient underwent several esophagogastric cauterization procedures but not positive outcomes were obtained, so he had to go to the Emergency Service of the hospital multiple times due to having low levels of hemoglobin (as low as 3.5g/dL). A bevacizumab based treatment was started by using a novel dosage regimen consisting of the administration of 6 5mg/kg bevacizumab dosages every 14 days. During the first week of treatment, hemoglobin levels increased to 14g/dL and the condition was stabilized. Conclusions: The findings reported here suggest that bevacizumab may be a therapeutic choice to be considered when treating patients with recurrent and refractory GI bleeding caused by HHT. However, a larger sample is required to determine if administering this medication is safe for these patients, as well as the appropriate dosage.

Resumen Introducción. La telangiectasia hemorrágica hereditaria (HHT) es una enfermedad genética autosómica dominante que se caracteriza por la presencia de malformaciones arteriovenosas en mucosa nasal, dedos y, algunas veces, pulmones, tracto gastrointestinal, hígado, páncreas, médula ósea y cerebro. El tratamiento se basa en el control sintomático, pero recientemente se ha propuesto la administración de moléculas anti-factor de crecimiento de endotelio vascular (VEGF), en especial en pacientes que presentan sangrado recurrente. Presentación del caso. Paciente masculino de 67 años con diagnóstico de telangiectasia hemorrágica hereditaria (HHT) y hemorragia gastrointestinal severa potencialmente mortal. El paciente recibió múltiples cauterizaciones esofagogástricas sin obtener respuesta, por lo que ingresó en múltiples oportunidades al servicio de urgencias con niveles de hemoglobina incluso tan bajos como 3.5g/dL. Se inició tratamiento con bevacizumab con un novedoso esquema de 6 dosis de 5mg/k cada 14 días, lográndose aumentar los niveles de hemoglobina a 14g/dL durante la primera semana de tratamiento y estabilizando la enfermedad. Conclusiones. Los hallazgos sugieren que el bevacizumab puede ser una opción terapéutica en sangrado gastrointestinal recurrente y refractario secundario a HHT. Sin embargo, se requiere incluir una cohorte de pacientes más amplia para establecer la seguridad del medicamento y la dosificación apropiada para este tipo de pacientes.

Detección del gen fusión PML-RARα en pacientes colombianos con leucemia mieloide aguda / Detection of the PML-RARα fusion gene in Colombian patients with acute myeloid leukemia

Resumen Introducción: la leucemia promielocítica aguda es un subtipo de leucemia mieloide aguda caracterizada por la presencia de una translocación entre los cromosomas 15 y 17 que provoca la formación de un gen fusión denominado PML/RARα. Determinar la presencia de este gen fusión es crítico para estos pacientes ya que su presencia hace el diagnóstico de la enfermedad, aún sin tener resultados de patología. Con esta investigación se busca ajustar e implementar una prueba altamente sensible y específica para la detección del reordenamiento PML/RARα. Métodos: a partir de sangre periférica se extrajo RNA de pacientes diagnosticados con leucemia mieloide aguda en dos instituciones de Antioquia (Colombia). Se realizó RT-PCR anidada para la detección de PML/RARα, ajustando un protocolo previamente publicado. Resultados: se ajustó y estandarizó un método para detectar mediante RT-PCR el gen fusión PML/RARα. Mediante esta técnica se logró identificar la traslocación en cuatro pacientes (22 %) de la cohorte estudiada. Conclusiones: los resultados están de acuerdo con estudios previos. La detección de esta y otras alteraciones citogenéticas mediante pruebas moleculares permitirá tener información valiosa a nivel de diagnóstico y pronóstico de los pacientes con leucemia mieloide aguda en Antioquia.

Abstract Introduction: acute promyelocytic leukemia is a subtype of acute myeloid leukemia characterized by the presence of a translocation between chromosomes 15 and 17, which causes the formation of a fusion gene called PML/RARα. Determining the presence of this fusion gene is critical for these patients, since their presence makes the diagnosis of the disease, even with no pathology results This research seeks to adjust and implement a highly sensitive and specific test for the diagnosis of this cytogenetic abnormality. Methods: peripheral blood samples from patients diagnosed with acute myelocytic leukemia were collected in two institutions of Antioquia (Colombia), from which RNA was extracted and nested RT-PCR was performed, adjusting a previously published protocol. Results: we adjusted and standardized a method to detect the PML/RARα fusion gene by RT-PCR. Using this technique, translocation was identified in four patients (22%) of the studied cohort. Conclusions: our results agree with previous studies. The detection of this and other cytogenetic alterations by means of molecular tests will allow to have valuable information at the level of diagnosis and prognosis of patients with AML in Antioquia.

Molecular biomarkers in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The pathophysiology of this disease is just beginning to be understood at the cellular and molecular level, and currently cytogenetic markers are the most important for risk stratification and treatment of AML patients. However, with the advent of new technologies, the detection of other molecular markers such as point mutations and characterization of epigenetic and proteomic profiles, have begun to play an important role in how the disease is approached. Recent evidence shows that the identification of new AML biomarkers contributes to a better understanding of the molecular basis of the disease, is significantly useful in screening, diagnosis, prognosis and monitoring of AML, as well as the possibility of predicting each individual's response to treatment. This review summarizes the most relevant molecular (genetic, epigenetic, and protein) biomarkers associated with acute myeloid leukemia and discusses their clinical importance in terms of risk prediction, diagnosis and prognosis.

Characterization of a murine neuron-enriched model of primary trigeminal ganglia cultures to study the interferon-ß antiviral effect against Herpes Simplex Virus type 1 / Caracterización de un modelo murino de cultivos primarios de ganglio trigeminal enriquecido en neuronas para el estudio del efecto antiviral del interferón-ß frente al virus Herpes simple tipo 1

Objective: To characterize a neuron-enriched primary TG culture and evaluate interferon- β expression and activity after HSV-1 infection. Materials and methods: The percentage of neurons present in cultures was assessed by neurofilament immunocytochemistry. Cultures were treated with interferon- β and infected with HSV-1, then viral antigen positive cells were counted and interferon- βexpression was assessed by quantitative PCR. Results: The culture contained 15% neurons and 85% non-neuronal cells. A cytopathic effect was observed, associated with high viral spread (72.9% neurons and 48.3% non-neuronal cells were positive for viral antigen). Interferon- β treatment impaired the cytopathic effect and decreased the infected neurons to 16.7% and infected non-neuronal cells to 7.8%. Viral infection at 6 h postinfection significantly increased the interferon- β transcripts by 18.2 fold, while at 18 h postinfection Interferon pre-treatment in infected cultures increased interferon- β transcription by 3.7 fold. Discussion: This culture model contained 15% neurons, which is 10 times higher compared to other reported cultures, and non-neuronal cells comprised 85% of cells in this culture. All types of cells were found to be infected, which is similar to that reported during acute infections in vivo . Additionally, interferon- βdecreased the infected cells, avoiding the cytopathic effect, which is similar to that reported in swine TG cultures. Conclusions: A neuron-enriched primary TG model was characterized. Interferon- β treatment protected cells from cytopathic effects and viral spread, while viral infection up-regulated interferon- β expression. This result means that interferon- β exerts an important antiviral effect against HSV-1 in these cultures.

Objetivo: Caracterizar un cultivo primario de ganglio trigeminal (GT) enriquecido en neuronas y evaluar la expresión de interferón- y su actividad frente a la infección con Herpes simple tipo 1 (HSV-1). Materiales y métodos: El porcentaje de neuronas fue determinado por inmunocitoquímica para neurofilamento. Los cultivos fueron tratados con interferón- β e infectados con HSV-1, y se cuantificaron las células positivas para antígeno viral por inmunocitoquímica y la expresión de interferón- β por PCR cuantitativa. Resultados: El cultivo presentó un 15% de neuronas y 85% de células no neuronales. Se encontró efecto citopático, asociado a una alta diseminación de la infección (72,9% neuronas y 48,3% de células no neuronales positivas para antígeno viral). El interferón- β evitó la aparición de efecto citopático y disminuyó las células infectadas a 16,7% en neuronas y a 7,8% las células no neuronales. La infección viral incrementó la expresión de transcritos de interferón- β 18,2 veces a las 6 h de infección, mientras que a las 18 h post infección el tratamiento con interferón incrementó esta expresión 3,7 veces. Discusión: Los cultivos presentaron un 15% de neuronas, lo cual es 10 veces más que en otros cultivos reportados. Las células no neuronales representan el 85% de las células del cultivo, y se evidenció que todos los tipos de células se infectaron; similar a lo que ha sido reportado durante infecciones agudas in vivo . Adicionalmente, el interferón- β disminuyó el porcentaje de células infectadas y evitó la aparición de efecto citopático, similar a lo que ha sido reportado en cultivos de GT porcino. Conclusiones: Se caracterizó un modelo de cultivo primario de GT enriquecido en neuronas. Interferón- β protegió las células del efecto citopático y la diseminación viral mientras que la infección viral incrementó la expresión de interferón- β. Por lo tanto, el interferón- β ejerció un papel antiviral importante frente al HSV-1 en estos cultivos.

Study of interferon-ß antiviral activity against Herpes simplex virus type 1 in neuron-enriched trigeminal ganglia cultures.

Herpes simplex virus type 1 (HSV-1) causes a lytic infection in epithelial cells before being captured and moved via retrograde axonal transport to the nuclei of the sensory neurons of the trigeminal ganglion or dorsal root, where it establishes a latent infection. HSV-1 infection induces an antiviral response through the production of Beta Interferon (IFN-ß) in infected trigeminal ganglia. The aim of this work was to characterize the response induced by IFN-ß in neuron-enriched trigeminal ganglia primary cultures infected with HSV-1. An antiviral effect of IFN-ß in these cultures was observed, including reduced viral production and increased cell survival. In contrast, viral infection significantly decreased both double stranded RNA dependent protein kinase (Pkr) transcription and Jak-1 and Stat-1 phosphorylation, suggesting a possible HSV-1 immune evasion mechanism in trigeminal cells. Additionally, HSV-1 infection upregulated Suppressor of Cytokine Signaling-3 (Socs3) mRNA; upregulation of socs3 was inhibited in IFN-ß treated cultures. HSV-1 infection increased the number of Socs3 positive cells and modified the intracellular distribution of Socs3 protein, in infected cells. This neuron-enriched trigeminal ganglia culture model could be used to elucidate the HSV-1 viral cycle in sensory neurons and to study cellular antiviral responses and possible viral evasion mechanisms that underlie the choice between viral replication and latency.

Bases genéticas de la formación de fisuras labiales y/o palatinas en humanos / Genetic basis of orofacial cleft formation in humans

La hendidura labio palatina (cleft lip/palate, CL/P) es una alteración craneofacial de alta frecuencia en la población mundial, cuya etiología es multifactorial, y que involucra factores tanto genéticos como ambientales. La mayoría de este tipo de patologías se presenta como un defecto no sindrómico (70%), y el resto de hendiduras se asocian con alteraciones adicionales en los defectos de tipo sindrómico. Gracias al desarrollo de herramientas moleculares se han identificado varios genes que están involucrados en CL/P de tipo no sindrómico, como son el proto-oncogen Bcl3, el gen Tgfb, el gen homeótico Msx1 y el gen Bmp entre otros, los cuales se han podido identificar mediante estudios de ligamiento y el uso de ratones deficientes en estos genes (knock-out). Por otra parte, se han identificado más de 300 síndromes diferentes asociados con CL/P, entre ellos uno de los más frecuentes el síndrome de van der Woude donde también se han identificado mutaciones en genes como el IRF6. Debido a que CL/P es una patología de alta complejidad etiológica donde las alteraciones genéticas juegan un importante papel y donde cada día se amplía el conocimiento sobre alteraciones en diversos genes que contribuyen a la formación de la alteración, el objetivo de este artículo, es revisar la información actualizada sobre la genética del CL/P y los genes reportados que pueden contribuir al desarrollo de esta compleja patología.

Clefts of the lip and palate (CL/P) are a craneofacial alteration of high frequency in the world-wide population, whose etiology is multifactorial, involving genetic factors as much as environmental ones. Most of this type of pathologies (70%) appear as non-syndromic form, although the presence of additional facial alterations is associated with clefts of syndromic type. The development of molecular tools has permitted to identify some of the genes that are involved in non-syndromic CL/P, such as proto-oncogen Bcl3, gene Tgfb, homeotic gene Msx1 and Bmp among others, which have demonstrated a relationships between them using both linkage analysis and knock-out mice. In the other hand, they have been identified more of 300 different syndromes associated with CL/P, being one from most frequent the van der Woude syndrome which also have identified mutations in genes such as IRF6. Because CL / P is a highly complex etiology pathology where genetic alterations play an important role and where every day is more knowledge on alterations in several genes that contribute to the formation of the alteration, the aim of this article is review the current knowledge on the reported genetics of the CL/P and genes that would contribute to the development of this complex pathology.

Comparative evaluation of permissiveness to dengue virus serotype 2 infection in primary rodent macrophages.

Infection with dengue virus presents a broad clinical spectrum, which can range from asymptomatic cases to severe cases that are characterised by haemorrhagic syndrome and/or shock. The reason for such variability remains unknown. This work evaluated the in vitro permissiveness of mouse, rat, hamster and guinea pig macrophages to infection by dengue virus 2 (DENV2). The results established that macrophages derived from the BALB/c mouse strain showed higher permissiveness to DENV2 infection than macrophages from other rodent species, although all rodent species studied had the C820T mutation in the oligoadenylate synthetase 1b gene, indicating no relationship to the different in vitro susceptibilities of mouse cells at this locus. Other molecular mechanisms related to flavivirus susceptibility remain to be explored.

Evaluación del valor diagnóstico de la detección de NS1 en pacientes con dengue agudo / Diagnostic value of NS1 detection in serum from acute dengue patients

El dengue es endemo-epidémico en Colombia y gran parte de nuestra población está en riesgo de padecer esta enfermedad. Uno de los principales problemas en el manejo del dengue es la dificultad para diagnosticar tempranamente esta arbovirosis, ya que la enfermedad presenta un cuadro clínico de evolución inespecífica, por lo cual es indispensable contar con herramientas diagnósticas rápidas y efectivas. El presente trabajo tiene como objetivo evaluar el valor diagnóstico de la detección de la proteína viral NS1 en pacientes con sospecha de dengue agudo, provenientes del Departamento de Cundinamarca, a quienes previamente se les había realizado detección de IgM en el Laboratorio de Salud Pública de Cundinamarca; de éstos, 44 muestras correspondían a pacientes con una prueba IgM positiva y las otras 44 a pacientes con una prueba de IgM negativa para dengue. A todas las muestras de suero se les practicó la prueba mediante el sistema Pan-E Dengue Early ELISA™ (Panbio) para la detección de NS1. Al hacer el análisis estadístico, se obtuvo una concordancia moderada entre las dos pruebas diagnósticas. Además, se encontró que 40 % de las muestras positivas para NS1, habían sido previamente reportadas como negativas de acuerdo con el resultado de IgM. De igual forma, se encontró enfermedad grave a menor edad y diferencias significativas en la presencia de erupción cutánea, ausencia de diarrea, leucocitos de menos de 4.000 células/μl y menos de 180.000 plaquetas/μl, al comparar los resultados positivos y negativos de cada prueba. Se puede concluir que la determinación de NS1 es necesaria en los casos sospechosos de dengue agudo y, junto con la determinación de IgM, representa un complemento eficiente para el diagnóstico, agilizando la instauración de un tratamiento oportuno.

Dengue is an endemic disease in Colombia and a large part of our population is at risk of suffering from this illness. One of the main problems in the management of dengue is the difficulty to do an appropriate and early diagnosis, because the disease presents an inespecific evolution, thus, it is essential to account on fast and effective diagnostic tools. The present work aimed to evaluate the diagnostic value of detection of the viral protein NS1 in patients with suspected acute dengue, from the Department de Cundinamarca, who had previously been submitted to IgM detection in the Cundinamarca Public Health Laboratory; 44 samples came from patients with a positive IgM test and the other 44 to patients with negative IgM for dengue virus. All serum samples were submitted to Pan Dengue Early ELISA (Panbio) system for NS1 detection. The statistical analysis showed a moderate concordance between the two diagnostic tests. We also found that 40% of positive samples for NS1 had previously been reported as negative according to the results of IgM. Simi1arly, it was found that as younger age, severe disease was present, there were found significant differences in the presence of rash, absence of diarrhea, leukocytes <4000 cel/μl and platelets <180,000/μl when comparing positive and negative results of each test. We can conclude that NS1 determination is needed in suspected cases of acute dengue, and in conjunction with the determination of IgM represents an efficient complement to diagnosis, thus expediting the implementation of early treatment.

La infección por virus dengue induce la disminución de marcadores de diferenciación en células de neuroblastoma / Dengue virus infection down-regulates differentiation markers in neuroblastoma cells

Introducción: cerca del 5% de los pacientes con dengue hemorrágico pueden presentar manifestaciones neurológicas; sin embargo, existe poca información sobre la infección directa por el virus dengue (DENV) en neuronas. Objetivo: determinar el papel del fenotipo neuronal en la infección por DENV en células de neuroblastoma SH-SY5Y inducidas o no a la diferenciación con ácido retinoico (AR). Materiales y métodos: células SH-SY5Y fueron inducidas con AR a diferenciarse e infectadas con DENV. Posteriormente se cuantificó la expresión de antígeno viral y de dos marcadores de diferenciación (GAP43 y sinaptofisina). También se evaluó la viabilidad postinfección por la técnica de MTT. Resultados: se encontró que las células diferenciadas son más susceptibles a la infección por DENV, pues se detectó en ellas mayor cantidad de antígeno viral que en las indiferenciadas. A pesar de que el virus indujo muerte celular en ambos tipos de células, la proporción fue mayor en las indiferenciadas (40,3% frente a 21,5%). La infección por DENV en células SH-SY5Y diferenciadas indujo una disminución significativa en la expresión de GAP-43 y sinaptofisina. Conclusiones: los resultados que se presentan permiten sugerir una relación entre la infección viral y la función neuronal, que podría ser importante para esclarecer la patogénesis de las manifestaciones neurológicas durante las formas graves de dengue.

Introduction: Approximately 5% of patients suffering from dengue hemorrhagic fever may have neurological manifestations. However, little information is available about direct infection of neurones by dengue virus. Objective: To determine the role of neuronal phenotype during DENV infection in human neuroblastoma cell line SH-SY5Y, either induced or not to differentiate by treatment with retinoic acid (RA). Materials and methods: Neuroblastoma cell line SH-SY5Y was induced to differentiate with RA and infected with DENV. The expression of viral antigen and of two differentiation markers of neurones, GAP-43 and synaptophysin, was evaluated quantitatively. Postinfection viability was also evaluated by the MTT technique. Results: It was found that differentiated cells are more susceptible to infection by dengue virus since more viral antigen was found in them than in the undifferentiated ones. DENV infection caused death in both cell types, but the rate was higher in the undifferentiated ones (40.3% vs 21.5%). In addition, DENV infection in differentiated SH-SY5Y cells induced a significant decrease in GAP-43 and synaptophysin expression. Conclusions: These results allow us to suggest a relationship between DENV infection and neuronal function, which could be important to elucidate the pathogenesis of neurological manifestations occurring in severe dengue disease.

Real time PCR: application in dengue studies: [review] / PCR en tiempo real: modelo de aplicación en dengue: [revisión]

PCR (polymerase chain reaction) is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.

La reacción en cadena de la polimerasa (PCR) es una herramienta usada de rutina en todos los laboratorios de diagnóstico e investigación. Esta técnica se utiliza en detección de mutaciones y patógenos, investigación forense, e incluso es la base para la secuenciación del genoma humano. Una modificación de la PCR, la PCR en tiempo real, permite la cuantificación de ácidos nucléicos con mayor sensibilidad, especificidad y reproducibilidad. Este artículo pretende revisar los principios de PCR en tiempo real y exponer un modelo de aplicación en virología en el que se cuantificó el número de copias virales producido a partir de macrófagos murinos infectados con virus dengue 2; los resultados obtenidos establecieron que la cepa murina BALB/c presenta una mayor susceptibilidad a la infección por virus dengue, lo que permite establecer esta cepa de ratones como un mejor modelo de estudio para la investigación de la fisiopatología de la infección por virus dengue.