Prediction of potential cysteine synthase inhibitors of Leishmania braziliensis and Leishmania major parasites by computational screening.

Leishmaniasis is a neglected tropical disease considered a public health problem that requires innovative strategies for its chemotherapeutic control. In the present investigation, a molecular docking approach was carried out using the protein cysteine synthase (CS) of Leishmania braziliensis (CSLb) and Leishmania major (CSLm) parasites to identify new compounds as potential candidates for the development of selective leishmaniasis therapy. CS protein sequence similarity, active site, structural modeling, molecular docking, and ADMET properties of compounds were analyzed using bioinformatics tools. Molecular docking analyses identified 1000 ligands with highly promising binding affinity scores for both CS proteins. A total of 182 compounds for CSLb and 173 for CSLm were selected for more detailed characterization based on the binding energy and frequency values and ADMET properties. Based on Principal Component Analysis (PCA) and K-means clusterization for both CS proteins, we classified compounds into 5 clusters for CSLb and 7 for CSLm, thus providing an excellent starting point for verification of enzyme inhibition in in vitro studies. We found the ZINC16524774 compound predicted to have a high affinity and stability for both CSLb and CSLm proteins, which was also evaluated through molecular dynamics simulations. Compounds within each of the five clusters also displayed pharmacological and structural properties that make them attractive drug candidates for the development of selective cutaneous leishmaniasis chemotherapy.

Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species.

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases' participation in protein-protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

Test accuracy of polymerase chain reaction methods against conventional diagnostic techniques for Cutaneous Leishmaniasis (CL) in patients with clinical or epidemiological suspicion of CL: Systematic review and meta-analysis.

BACKGROUND: Molecular diagnostic tests, notably polymerase chain reaction (PCR), are highly sensitive test for Leishmania detection, which is especially relevant in chronic cutaneous lesion with lower parasite load. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. Nevertheless, diagnosis of cutaneous leishmaniasis (CL) is hampered by the absence of a reference standard. Assuming that the PCR-based molecular tools are the most accurate diagnostic method, the objective of this systematic review was to assess the diagnostic accuracy of PCR-based molecular tools in a meta-analysis of the published literature. METHODOLOGY/PRINCIPAL FINDINGS: A search of the published literature found 142 papers of which only 13 studies met the selection criteria, including conventional PCR, real-time PCR, Loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), polymorphism-specific PCR (PS-PCR). The sensitivities of the individual studies ranged from 61% to 100%, and specificities ranged from 11% to 100%. The pooled sensitivities of PCR in smears were 0.95 (95% CI, 0.90 to 0.98), and the specificity was 0.91(95% CI, 0.70 to 0.98). In general population, estimates were lower in aspirates, skin biopsies and swab samples with 0.90 (95% CI, 0.80 to 0.95) and 0.87 (95% CI, 0.76 to 0.94) for sensitivity and specificity, respectively. The specificity was lower in consecutive studies, at 0.88 (95% CI, 0.59 to 0.98) and its CI were wider. CONCLUSIONS/SIGNIFICANCE: No statistically significant differences between the accuracy in smears, aspirate, skin biopsies or swabs samples were observed. Therefore, a simple smear sample run by PCR, instead more invasive samples, may be enough to obtain a positive diagnosis of CL. The results for PCR in all samples type confirm previous reports that consider PCR as the most accurate method for the diagnosis of CL.

Morphological and molecular characterization of Paragonimus caliensis Little, 1968 (Trematoda: Paragonimidae) from Medellin and Pichinde, Colombia.

Paragonimiasis is a subacute to chronic inflammatory granulomatous lung disease caused by the genus Paragonimus. In Latin America Paragonimus mexicanus Miyazaki & Ishii, 1968 is the only confirmed species to cause human infections. Paragonimus caliensis Little, 1968 is an uncommon species often regarded as a synonym of P. mexicanus. Recently, the study of two types of Paragonimus metacercariae from Costa Rica has provided new molecular and morphological evidence that P. caliensis is a separate species from P. mexicanus. In the present study, molecular, morphological and phylogenetic tools have been used to characterize two populations of Paragonimus located at west of Medellin, Antioquia and at Pichinde, Valle del Cauca (type locality of P. caliensis), Colombia. Adults and metacercariae obtained from Medellin, and metacercariae from Pichinde were analyzed. For morphological observations we used light microscopy and scanning electron microscopy (SEM). Morphology of metacercariae and adults matched with the holotype of P. caliensis. The number and arrangement of sensory papillae in the acetabulum region differs from the morphotypes reported for P. caliensis in Costa Rica. Two morphotypes in branching patterns of ovary and two morphotypes in branching patterns of testes were identified. The main morphological differences between P. caliensis and P. mexicanus corresponded to the size of gonads and their relative positions in the body, and the occasional presence of a cyst wall in P. caliensis metacercariae. The molecular and phylogenetic analyses (using nuclear ribosomal ITS2 and partial cytochrome c oxidase subunit 1 CO1 sequences) confirmed that P. caliensis from the type locality is the same species from Medellin and Costa Rica. Furthermore, these analyses also suggest genetic as well as geographical separation of P. caliensis populations between Colombia and Costa Rica. Currently, P. mexicanus and P. caliensis are sympatric in the Colombian Pacific bioregion, and specific diagnosis based on their egg size is not possible. Therefore, it is necessary to determine the biogeographic distribution ranges of both species and to implement molecular techniques to establish the role of P. caliensis in human paragonimiasis in Colombia.

Detección de mutaciones en el dominio tirosina quinasa de BCR-ABL1 en pacientes colombianos con leucemia mieloide crónica LMC, resistentes al imatinib / Detection of BCR-ABL1 tyrosine kinase domain mutations in chronic myeloid leukaemia patients with Imatinib resistance in Colombia

Resumen Introducción: Las mutaciones en el dominio BCR-ABL1, tirosina quinasa (TK) son mecanismos importantes de resistencia de los inhibidores de la tirosina quinasa (ITK) en pacientes con leucemia mieloide crónica (LMC). Objetivo: Determinar el tipo y la frecuencia de las mutaciones en el dominio tirosina quinasa del gen BCR-ABL1, asociadas con falla en la respuesta al tratamiento con imatinib en pacientes con LMC y correlacionar el perfil de mutaciones con los hallazgos clínicos, demográficos, respuesta citogenética y respuesta molecular. Materiales y métodos: Se realizó un estudio descriptivo de tipo prospectivo en pacientes con LMC en tratamiento con IMATINIB a quienes se les realizó cariotipo y análisis de mutaciones del dominio BCR-ABL1 mediante la técnica de PCR anidada. Resultados: De los 23 pacientes estudiados en cuatro se encontraron mutaciones: dos presentaron la mutación E255K, uno presentó la mutación H396P y otro presentó doble mutación L387L y T389P. Las mutaciones E255K que se ubican en la región P-loop y H396P en A-loop se asocian con mal pronóstico. La mutación T389P localizada en la región A-loop no está informada en algunas bases de datos. Conclusiones: En este estudio encontramos cuatro mutaciones en el dominio tirosina quinasa (E255K, H396P, L387L y T389P) que podrían aportar información valiosa y guiar las decisiones de tratamiento. Es importante destacar que esta investigación de análisis mutacional del dominio BCR-ABL es la primera que se realiza en el país con la particularidad adicional de cubrir una población triétnica.

Abstract Mutations in the BCR-ABL1 tyrosine kinase domain mutations, are one of the principal mechanisms associated with tyrosine kinase inhibitors (TKI) resistance in patients with chronic myeloid leukaemia (CML). Objectives: To determine the type and frequency of mutations in the tyrosine kinase domain of the BCR-ABL1 gene associated with failure to respond to treatment with Imatinib and Imatinib in patients with CMK, and to correlate the mutation profile with the clinical and demographic variables, as well as the cytogenetic and molecular response. Materials and methods: A descriptive prospective study was carried out on patients with CML treated with Imatinib. Karyotyping and analysis of the BCR-ABL1 domain mutations were performed on the patients using nested PCR. Results: Four types of mutations were found in the 23 patients studied, of which two of them were the E225 mutation, one with the H396P mutation, another with a double mutation L387L and T389P. Both the E255K mutation located in the P-loop region, and H396P mutation in the A-loop region, are associated with a poor prognosis. The T389P mutation located within A-loop region has not been reported in any of the databases. Conclusions: Four mutations were found in the tyrosine kinase domain (E255K, H396P, L387L and T389P) were found in this study. These findings provide valuable information and as a guideline to help make treatment decisions. It is important to point out that this analytical study on mutations of the BCR-ABL domain is the first one carried out in the country and, specifically, in a tri-ethnic population.

Curvas de fusión de regiones genómicas específicas: una herramienta prometedora para el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia / High-resolution melting analysis based on specific genomic regions: A promising tool for the diagnosis and typing of species causing cutaneous leishmaniasis in Colombia

Resumen Introducción. La leishmaniasis cutánea es una enfermedad causada por parásitos del género Leishmania que tiene gran incidencia en Colombia. El diagnóstico y la identificación de la especie infecciosa son factores críticos en el momento de escoger e iniciar el tratamiento. Actualmente, los métodos de diagnóstico y tipificación requieren procedimientos complejos, por lo que es necesario validar nuevos marcadores moleculares y métodos que simplifiquen el proceso. Objetivo. Desarrollar una herramienta basada en la reacción en cadena de la polimerasa (PCR) con curvas de fusión (High Resolution Melting; PCR-HRM) para el diagnóstico y tipificación de las tres especies de Leishmania de importancia epidemiológica en casos de leishmaniasis cutánea en Colombia. Materiales y métodos. Los genomas de Leishmania panamensis, L. braziliensis y L. guyanensis se compararon mediante métodos bioinformáticos. Las regiones específicas de especie identificadas se validaron mediante PCR. Para los marcadores seleccionados se diseñó una PCR-HRM y se estimaron algunos parámetros de validez y seguridad usando aislamientos de pacientes colombianos caracterizados previamente mediante PCR y análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism - RFLP; PCR-RFLP) del gen hsp70. Resultados. El análisis genómico comparativo mostró 24 regiones específicas de especie. Sin embargo, la validación mediante PCR solo identificó un marcador específico para cada especie de Leishmania. Los otros marcadores mostraron amplificación cruzada. El límite de detección para los tres marcadores seleccionados fue de un parásito, mientras que la sensibilidad, la especificidad, el valor predictivo positivo y el negativo fueron de 91,4, 100, 100 y 75 %, respectivamente. Conclusiones. Las tres regiones seleccionadas pueden emplearse como marcadores moleculares en el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia.

Abstract Introduction: Cutaneous leishmaniasis, caused by parasites of the genus Leishmania, is a disease with high incidence in Colombia. The diagnosis and identification of the infectious species are critical factors when selecting and initiating treatment. Currently, the methods for diagnosing and typing cutaneous leishmaniasis require complicated procedures and there is a need for the validation of new molecular markers and methods to simplify the process. Objective: To develop a tool based in PCR melting curves (PCR-HRM) for the diagnosis and typing of the three Leishmania species of epidemiological importance for cutaneous leishmaniasis in Colombia. Materials and methods: The genomes of Leishmania panamensis, L. braziliensis and L. guyanensis were compared with bioinformatic methods. The species-specific regions were then validated using PCR. For the selected markers, a PCR-HRM was designed, and validity and security parameters were estimated using isolates from Colombian patients previously characterized by PCR-RFLP of the hsp70 gene. Results: The comparative genomic analysis yielded 24 species-specific regions. However, the PCR validation identified only one marker that was specific to each Leishmania species. The other markers showed cross amplification. The detection limit for the three selected markers was one parasite. The sensitivity, specificity, predictive positive and negative values were 91.4%, 100%, 100% and 75%, respectively. Conclusions: The three selected regions can be used as molecular markers in the diagnosis and typing of the causative species of cutaneous leishmaniasis in Colombia.

[Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia].

INTRODUCTION: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. OBJECTIVE: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). MATERIALS AND METHODS: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. RESULTS: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. CONCLUSIONS: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

BACKGROUND: Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. RESULTS: We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. CONCLUSIONS: Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and therefore could become a novel therapeutic and druggable target in leishmaniasis therapy. In addition, following comparative sequence analyses, we found the RAC/AKT-like proteins from Leishmania constitute a subgroup by themselves within a general AKT-like protein family.

Efecto mutagénico y genotóxico, y expresión de los genes Rad51C , Xiap , P53 y Nrf2 inducidos por extractos antipalúdicos de plantas recolectadas en el Vaupés medio, Colombia / Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia

Resumen Introducción. Dada la resistencia de Plasmodium a los medicamentos antipalúdicos, es necesario encontrar nuevas alternativas terapéuticas para su tratamiento y control. Con base en el saber indígena colombiano, se recopilaron extractos de plantas del Vaupés medio con potencial efecto antipalúdico. Objetivo. Evaluar el efecto mutagénico y genotóxico, y la expresión de los genes Rad51C, Xiap, P53 yNrf2, inducidos por cuatro extractos etanólicos con actividad anti-Plasmodium(R001, T002, T015 y T028). Materiales y métodos. Se evaluó el potencial mutagénico de cuatro extractos etanólicos con efecto antiplasmódico utilizando el test de Ames y el efecto genotóxico, con un ensayo del cometa; asimismo, se analizó la expresión de los genes Rad51C, Xiap, P53 y Nrf2 en células HepG2. Resultados. Los extractos no fueron mutágenos en la cepa TA98 de Salmonella typhimurium en presencia y ausencia de actividad metabólica de la fracción S9. En la cepa TA100, los extractos R001, T015 y T028 se comportaron como mutágenos débiles en presencia de S9, con índices mutagénicos de 1,58; 1,38; 1,53 y 1,61, respectivamente; T015 tuvo el mismo comportamiento en ausencia de S9, con un índice mutagénico de 1,36. En el ensayo del cometa, todos los extractos provocaron daño de categorías 1 o 2, con colas de cometas entre 36,7 y 51,48 µm de longitud; sin embargo, el índice dedaño genético sugirió que los tratamientos afectaron la mayoría de las células. En los genes en estudio, los extractos R001 y T028 indujeron una sobreexpresiónde 1,84 a 3,99 frente a las células sin tratar de los genes Xiap y P53. Conclusiones. Los resultados evidenciaron que el extracto T002 fue el más seguro, ya que presentó actividad anti-Plasmodium, no fue citotóxico en las células HepG2, no fue mutágeno, causó daño de categoría 1 en el ADN y no modificó la expresión de los genes evaluados.

Abstracts Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). Materials and methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Identificación molecular con base en el gen hsp 70 de aislamientos clínicos de Leishmania spp. en Colombia / Molecular identification of Leishmania spp. in clinical isolates from Colombia based on hsp 70 gene

Introducción. La leishmaniasis es una enfermedad de gran prevalencia en Colombia, donde al menos seis especies diferentes la originan en el ser humano, con un amplio rango de características clínicas. La tipificación de la especie es importante, no solo desde el punto de vista epidemiológico, sino para el diagnóstico, dado que el tratamiento puede variar dependiendo de la especie identificada. En la identificación se han utilizado varias alternativas metodológicas con diferentes niveles de poder discriminatorio. Objetivo. Identificar especies de Leishmania mediante la amplificación molecular de un fragmento del gen hsp 70. Materiales y métodos. Se amplificó un fragmento del gen hsp 70 mediante reacción en cadena de la polimerasa (PCR- hsp 70) y se hizo el análisis de los polimorfismos de la longitud ( Restriction Fragment Length Polymorphism, RFLP) de los fragmentos de restricción de 81 aislamientos clínicos de Leishmania spp. de pacientes con leishmaniasis cutánea y mucocutánea para la identificación de las especies presentes. Resultados. Se obtuvo un solo amplicón para todas las muestras analizadas. La restricción enzimática de los 81 productos permitió la identificación de 70 con dos patrones de bandas que correspondían a dos alelos de Leishmania braziliensis (62 y ocho aislamientos, respectivamente), nueve aislamientos compatibles con L. panamensis y dos con L. guyanensis . El origen geográfico de los aislamientos coincidió con el de reportes previos sobre la distribución de dichas especies en Colombia. Conclusiones. La técnica de la PCR- hsp 70 y el análisis de RFLP fueron útiles para identificar las especies de Leishmania aisladas de muestras clínicas de Colombia y pueden aplicarse también en el estudio de cepas de vectores y reservorios de importancia epidemiológica.

Introduction: Leishmaniasis is highly prevalent in Colombia, where at least six different species can cause disease of varying clinical presentations in humans. The identification of the infecting species is quite important for prognosis, therapeutics and epidemiology. Different techniques with variable discriminatory power have been used for the identification. Objective: To carry out the molecular identification of Leishmania species through the amplification of a fragment of the hsp 70 gene. Materials and methods: Molecular amplification of the hsp 70 gene fragment (PCR- hsp 70) followed by restriction fragment length polymorphism analysis (RFLP) was done for identification purposes using DNA from 81 clinical isolates of Leishmania . Results: A single amplicon was obtained for all samples analyzed. The enzymatic restrictions of the 81 PCR products identified 70 with a banding pattern corresponding to L. braziliensis with two different patterns (62 and eight isolates, respectively), nine isolates compatible with L. panamensis and two with L. guyanensis . The geographical origin of the isolates is consistent with previous reports about the distribution of the corresponding species in Colombia. Conclusions: The PCR- hsp 70/RFLP technique used is a valid tool for the identification of Leishmania species isolated from clinical samples of patients in Colombia, which may also be applicable to the study of strains obtained from vectors and reservoirs with epidemiological significance.

[Detection of molecular targets on the phosphatidylinositol signaling pathway of Leishmania spp. through bioinformatics tools and mathematical modeling]. / Detección de blancos moleculares en la ruta de señalización del fosfatidil-inositol en Leishmania spp. mediante herramientas bioinformáticas y de modelado matemático.

INTRODUCTION: Leishmaniasis is a disease of high impact on public health. Research on drugs for its treatment is considered a priority by the World Health Organization. The phosphatidyl-inositol signaling pathway is interesting to explore because it is involved in the survival of the parasite, by controlling osmoregulation, transport through membranes, and activation of transcription factors. OBJECTIVE: To propose drug targets against the disease through bioinformatic analysis and mathematical modeling of this signaling pathway. MATERIALS AND METHODS: The phosphatidyl-inositol pathway proteins were characterized through Pfam and TriTrypDB databases. Subsequently, a similarity analysis with human proteins was performed using the OrthoMCL and InParanoid7 tools. Finally, a boolean model of the pathway was proposed using PROMOT and CellNetAnalyzer softwares. RESULTS: The phosphatidyl-inositol signaling pathway in Leishmania spp. was reconstructed and described. The similarity analysis determined the feasibility of the phosphatidyl-inositol pathway proteins as molecular targets. Mathematical models allowed integrating the elements of the path and predicted an inhibitor effect. The following were proposed as drug targets: inositol-3-phosphate-5-phosphatase, phosphatidylinositol-4-kinase, phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and Inositol-1P-polyphosphate phosphatase. CONCLUSION: The phosphatidyl-inositol signaling pathway is robust from the point of view of the qualitative model and the proteins found. Thus, potential drug targets against leishmaniasis were identified. Subsequently we will seek to detect drugs against this set of proteins and validate them experimentally .

Detección de blancos moleculares en la ruta de señalización del fosfatidil-inositol en Leishmania spp. mediante herramientas bioinformáticas y de modelado matemático / Detection of molecular targets on the phosphatidylinositol signaling pathway of Leishmania spp. through bioinformatics tools and mathematical modeling

Introducción. La leishmaniasis es una enfermedad de gran impacto en la salud pública. La Organización Mundial de la Salud considera prioritaria la investigación orientada al desarrollo de medicamentos para su tratamiento. La exploración de la ruta del fosfatidil-inositol es interesante, ya que está implicada en la supervivencia del parásito mediante el control de la osmorregulación, el transporte a través de las membranas y la activación de diversos factores de transcripción. Objetivo. Proponer blancos para el desarrollo de medicamentos contra la leishmaniasis mediante el análisis bioinformático y el modelado matemático de esta ruta. Materiales y métodos. Se caracterizaron las proteínas pertenecientes a la ruta del fosfatidil-inositol en las bases de datos TriTrypDB y Pfam. Posteriormente, se hizo un análisis de similitud con las proteínas humanas mediante las herramientas InParanoid7 y OrthoMCL. Finalmente, se propuso un modelo booleano de la ruta, utilizando los programas PROMOT y CellNetAnalyzer. Resultados. Se reconstruyó y se describió la ruta de señalización del fosfatidil-inositol en Leishmania spp. El análisis de similitud con proteínas humanas determinó la viabilidad de las proteínas pertenecientes a la ruta del fosfatidil-inositol como potenciales blancos moleculares. Los modelos matemáticos permitieron integrar los elementos de la ruta y predecir un efecto inhibidor. Se propusieron los siguientes blancos para el desarrollo de medicamentos: inositol-3-fosfato-5-fosfatasa, fosfatidil-inositol-4-cinasa, fosfatidil-inositol-3,4,5-trisfosfato-3-fosfatasa, e inositol-polifosfato1P-fosfatasa. Conclusiones. La ruta de señalización del fosfatidil-inositol aparece como una alternativa sólida desde el punto de vista del modelo cualitativo y a partir de las proteínas encontradas. Se identificaron posibles blancos de medicamentos contra la leishmaniasis. Posteriormente, se buscarán medicamentos contra las proteínas detectadas y se hará la validación experimental.

Introduction: Leishmaniasis is a disease of high impact on public health. Research on drugs for its treatment is considered a priority by the World Health Organization. The phosphatidyl-inositol signaling pathway is interesting to explore because it is involved in the survival of the parasite, by controlling osmoregulation, transport through membranes, and activation of transcription factors. Objective: To propose drug targets against the disease through bioinformatic analysis and mathematical modeling of this signaling pathway. Materials and methods: The phosphatidyl-inositol pathway proteins were characterized through Pfam and TriTrypDB databases. Subsequently, a similarity analysis with human proteins was performed using the OrthoMCL and InParanoid7 tools. Finally, a boolean model of the pathway was proposed using PROMOT and CellNetAnalyzer softwares. Results: The phosphatidyl-inositol signaling pathway in Leishmania spp. was reconstructed and described. The similarity analysis determined the feasibility of the phosphatidyl-inositol pathway proteins as molecular targets. Mathematical models allowed integrating the elements of the path and predicted an inhibitor effect. The following were proposed as drug targets: inositol-3-phosphate-5-phosphatase, phosphatidylinositol-4-kinase, phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and Inositol-1P-polyphosphate phosphatase. Conclusion: The phosphatidyl-inositol signaling pathway is robust from the point of view of the qualitative model and the proteins found. Thus, potential drug targets against leishmaniasis were identified. Subsequently we will seek to detect drugs against this set of proteins and validate them experimentally .

Frecuencia de los transcriptos p190BCR-ABL y p210BCR-ABL en una población colombiana con leucemia mieloide crónica (LMC) usando RT-PCR cualitativa / Detection of p190BCR-ABL AND p210BCR-ABL fusion transcripts in patients with chronic myeloid leukemia (CML) using qualitative RT-PCR / Frequência dos transcritos p190BCR-ABL e p210BCR- ABL numa população colombiana com leucemia mielóide crônica (LMC) usando RT-PCR qualitativa

Introducción: la leucemia mieloide crónica (LMC) se caracteriza por la presencia del cromosoma Filadelfia (Ph) que resulta de la translocación recíproca balanceada t(9;22)(q34;q11); este marcador cromosómico se encuentra con menor frecuencia en pacientes con leucemia linfoide aguda (LLA). Objetivo: determinar la frecuencia de las fusiones génicas BCR-ABL, que codifican para los transcriptos p210BCR-ABL y p190 BCR-ABL en pacientes colombianos con diagnóstico de LMC, en diferentes fases de la enfermedad o de su tratamiento. Materiales y métodos: estudio descriptivo de corte transversal de 31 pacientes con LMC (15-78 años). El análisis se hizo a partir de muestras de sangre periférica con la técnica PCR anidada cualitativa para las isoformas P210 BCR-ABL (b3a2 e b2a2) y P190 BCR-ABL (e1a2). Resultados: se detectó el transcripto p210BCR-ABL en 29 de los 31 casos (93,6%). En ellos se identificaron las fusiones génicas b2a2 (16/29; 55,2%), b3a2 (10/29; 34,5%) y la coexpresión b3a2 y b2a2 (3/29; 10,3%). Conclusión: la fusión génica b2a2 fue la más frecuente en esta población con LMC.

Introduction: Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph), resulting from the balanced reciprocal translocation t(9;22)(q34;q11). This marker chromosome is found less frequently in patients suffering from acute lymphoblastic leukemia. Objective: To determine the frequency of BCR-ABL gene fusions encoding the p210BCR-ABL y p190 BCR-ABL transcripts in Colombian patients diagnosed with CML in different stages of the disease and/or its treatment. Materials and methods: Cross sectional, descriptive study of thirty one CML patients (aged 15-78). Analysis was carried out through qualitative nested PCR for the isoforms P210 BCR-ABL (b3a2 e b2a2) and P190 BCR-ABL (e1a2), and based on peripheral blood samples. Results: In 29 of the 31 patients (93.6%) transcript p210BCR-ABL was detected; b2a2 and b3a2 gene fusions and the coexpression b3a2 y b2a2 were identified in 55.2% (16/29), 34.5% (10/29) and 10.3% (3/29) of the cases, respectively. Conclusion: b2a2 gene fusion was the most frequent in this CML population.

Introdução: a leucemia mielóide crônica (LMC) caracteriza- se pela presença do cromossomo Filadélfia (Ph) que resulta da translocação recíproca balanceada t(9;22)(q34;q11); este marcador cromossômico se encontra com menor frequência em pacientes com leucemia linfoide aguda (LLA). Objetivo: determinar a frequência das fusões genéticas BCR-ABL, que codificam para os transcritos p210BCR-ABL e p190 BCR-ABL em pacientes colombianos com diagnóstico de LMC, em diferentes fases da doença ou de seu tratamento. Materiais e métodos: estudo descritivo de corte transversal de 31 pacientes com LMC (15-78 anos). A análise se fez a partir de mostras de sangue periférico com a técnica PCR aninhada qualitativa para as isoformas P210 BCR-ABL (b3a2 e b2a2) e P190 BCR-ABL (e1a2). Resultados: detectou-se o transcrito p210BCR-ABL em 29 dos 31 casos (93,6%). Neles se identificaram as fusões genéticas b2a2 (16/29; 55,2%), b3a2 (10/29; 34,5%) e a co-expressão b3a2 e b2a2 (3/29; 10,3%). Conclusão: a fusão genética b2a2 foi a mais frequente nesta população com LMC.

Detección de Leishmania spp. en base al gen que codifica la proteína HSP20 / Detection of Leishmania spp. based on the gene encoding HSP20

Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20) para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR).Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (ƙ=0,731), lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (Ƙ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.

Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania ( Viannia ) and its evaluation as a drug target / Caracterización bioquímica de la enzima bifuncional dihidrofolato reductasa-timidilato sintasa de Leishmania ( Viannia ) y su evaluación como blanco molecular

Introduction. Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania ( Viannia ) species . Materials and methods. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. Results. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed K m and V max values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H 2 F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity ( Ki = 22.0 µM) than trimethoprim ( Ki = 33 µM) and pyrimethamine ( Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. Conclusion. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Introducción. La dihidrofolato reductasa (DHFR) se ha utilizado como blanco molecular en tratamientos antibacterianos, anticancerígenos y antipalúdicos. También, actúa como blanco molecular en Leishmania ; sin embargo, no existen reportes de la enzima bifuncional en especies de Leishmania ( Viannia ). Materiales y métodos. Se ha aislado y expresado en Escherichia coli el gen que codifica para la enzima bifuncional DHFR y la timidilato-sintasa (TS) de Leishmania braziliensis . La enzima recombinante se purificó y caracterizó, y se evaluó el efecto inhibitorio de algunos antifolatos, así como de cuatro alcaloides aporfínicos. Resultados. El gen se compone de aproximadamente 1.560 pb y codifica un péptido de 58 kDa que contiene los dominios DHFR y TS ligados en una sola cadena polipeptídica. La enzima recombinante DHFR-TS, utilizando el dihidrofolato (H2F) como sustrato, presentó valores de K m y V max de 55,35 ± 4,02 (media ± el error estándar de la media) y de 0,02 ± 5,34 x 10 -4 , respectivamente. La enzima rDHFR-TS de L. braziliensis presentó valores de Ki para los antifolatos en el rango de micras. El metotrexato fue el inhibidor más potente de la actividad enzimática ( Ki =22,0 mM) en comparación del trimetoprim ( Ki =33 mM) y la pirimetamina ( Ki =68 mM). Estos valores de Ki son significativamente más bajos en comparación con los obtenidos para los alcaloides aporfínicos. Conclusión. Los resultados muestran el efecto inhibitorio de los antifolatos sobre la actividad enzimática, lo cual indica que la rDHFR-TS de L. braziliensis podría ser un modelo para estudiar moléculas antiprotozoarias potenciales.

Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania (Viannia) and its evaluation as a drug target.

INTRODUCTION: Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania (Viannia) species. MATERIALS AND METHODS: The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. RESULTS: The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed Km and Vmax values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H2F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity (Ki = 22.0 µM) than trimethoprim (Ki = 33 µM) and pyrimethamine (Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. CONCLUSION: The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Identification of Brucella canis Group 2 in colombian kennels / Identificación de Brucella canis Grupo 2 en criaderos de perros en Colombia / Identificação de Brucella canis Grupo 2 em criadouros de cães na Colômbia

Background: recently, isolates of Brucella canis (B. canis ) were classified into two groups, Group 1 and Group 2, based on a 794 bp deletion in the polysaccharide deacetylase gene in Group 1, or absence of this deletion in Group 2. In Colombia, B. canis was first isolated from sick or clinically healthy seropositive dogs in 2005. Objective: to determine Brucella canis genotype in isolates from Medellín Metropolitan Area. Methods: 49 isolates from a study involving 193 dogs in 10 kennels were analyzed by Polymerase Chain Reaction (PCR) using three primer pairs of eight previously reported. Results: all isolates were positive for 152, 272, and 794 bp fragments, indicating that they belong to Group 2. Conclusion: this article presents strong evidence based on the new molecular marker used to classify B. canis isolates from Medellín (Colombia) into Group 2. This is a pioneer molecular study on the presence of B. canis in Colombia and it would be interesting to apply this method to characterize the infection epidemiology and explore its domestic clinical dynamics.

Antecedentes: recientemente, las cepas de Brucella canis (B. canis ) fueron clasificadas dentro de dos grupos, Grupo 1 y Grupo 2, de acuerdo a la presencia o ausencia de una deleción de 794 bp en el gen de la polisacárido deacetilasa, respectivamente. En Colombia Brucella canis se aisló por primera vez en 2005 de perros seropositivos, enfermos o clínicamente sanos. Objetivo: determinar el genotipo de aislamientos de Brucella canis provenientes del Area Metropolitana de Medellín. Método: 49 aislamientos de un estudio con 193 perros en 10 criaderos fueron sometidos a tres Reacciones en Cadena de la Polimerasa (PCR) simple, empleando 3 pares de cebadores de 8 previamente reportados. Resultados: todos los aislamientos amplificaron los fragmentos de 152, 272 y 794 bp, indicando que pertenecen al Grupo 2. Conclusiones: este artículo presenta evidencia sólida para clasificar las cepas de Brucella canis aisladas en Medellín (Colombia) en el Grupo 2, basado en el uso del nuevo marcador molecular. Este es un estudio molecular pionero de B. canis en Colombia y sería interesante aplicar este método para caracterizar la dinámica epidemiológica y clínica de la infección en el país.

Antecedentes: atualmente, as cepas de Brucella canis foram classificadas em dois grupos, chamados de grupo 1 e 2, de acordo com a presença de uma deleção de 794 pb em seu genoma. No ano 2005, isolaram-se a Brucella canis pela primeira vez na Colômbia, obtida de cães doentes e soropositivos. Neste artigo descrevem-se a classificação de cepas de B. canis isolados em Medellín (Colômbia), de acordo com a nova norma. Objetivo: determinar o genotipo de isolamentos de Brucella canis a partir de amostras da Área Metropolitana de Medellín. Métodos: neste estudo utilizaram-se 49 isolamentos obtidos de 193 cães de 10 criadouros diferentes, os quais foram submetidos para três analises de Reação em Cadeia da Polimerase PCR simples com a utilização de três pares primers dos anteriormente informados. Resultados: todos os isolamentos analisados amplificaram fragmentos de 152, 272 e 749 pb, comprovando que pertencem ao grupo 2. Conclusões: este artigo apresenta evidencia solida para clasificar as cepas de Brucella canis isoladas em Medellín (Colombia) no grupo 2, baseados no uso do novo marcador molecular. Seria de grande importância que os estudos de epidemiologia molecular ampliarem-se para outras áreas de pesquisa na Colômbia, com a finalidade de seguir os patrões das infecções e fazer as correlações clinicas.

Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina / Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate.

Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento, se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre las proteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia, como Leishmania panamensis. Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis (UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínas en mecanismos de resistencia. Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, se compararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueron aisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas se cuantificó mediante PCR en tiempo real. Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43 manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente, identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica), isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α del proteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25). Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalente en L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismo de resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estas proteínas como blancos terapéuticos.

Introduction. The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. Objective. Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. Materials and methods. The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. Results. On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). Conclusion. This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites.

BACKGROUND: The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. CONCLUSIONS/SIGNIFICANCE: Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.