Differential gut microbiome in spondyloarthritis patients associated to Blastocystis colonization.

The role of Blastocystis in intestinal health is an open controversy, and little is known about the potential effect of this microorganism in autoinflammatory diseases such as spondyloarthritis (SpA). Here, we analyzed the gut microbiome of 36 SpA patients and 13 control individuals and demonstrated that the richness, diversity, and taxonomic composition between these two groups are different. We also showed that colonization by Blastocystis in control individuals increases the richness and diversity of the intestinal microbiome, whereas in SpA patients, it does not seem to have any impact. This may reflect a potential role of Blastocystis in sculpting the gut microbiome architecture in control individuals, whereas in subjects with SpA, the modulation of the microbiome may be governed by disease-dependent factors that cannot be overcome by Blastocystis. Regarding taxonomic characterization, SpA patients colonized by Blastocystis showed significant increases in the phylum Pseudomonadota, class Gammaproteobacteria, family Succinivibrionaceae, and genus Succinivibrio. Simultaneously, there were significant increases in the class Bacilli, order Lactobacillales, families Lactobacillaceae and Clostridiaceae, and genera Lactobacillus and Clostridium in non-colonized SpA patients. On the other hand, PICRUSt analysis in Blastocystis-positive SpA patients showed elevations in pathways that may enhance antioxidant capacities and alleviate intestinal inflammation, while Blastocystis-negative SpA patients showed significant changes in pathways that promote cell division/proliferation and can lead to larger changes in the gut microbiome. Our analyses lead us to believe that these changes in the gut microbiome of SpA patients may trigger protective mechanisms as an initial response to inflammation in an attempt to restore balance in the intestinal environment.

Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk.

Blastocystis is a common intestinal protist with a global distribution in humans and many other animals. Yet, the status of Blastocystis as a pathogen, the risk factors associated with its transmission, and its zoonotic potential remain ill-defined. Here, we explored subtype (ST) diversity and potential risk factors for Blastocystis infection in 98 children from Apulo, Colombia. Samples were screened for Blastocystis via PCR, and ST identification was performed through next-generation amplicon sequencing (NGS). Associations between the presence of Blastocystis and individual STs and sociodemographic variables were assessed via logistic regression analyses. Seventy-one samples (72.4%) were Blastocystis-positive, and NGS revealed the presence of five STs (ST1-ST5). ST1, ST2, and ST3 were common and observed in nearly equal proportions (~ 40%), while samples with ST4 (1.4%) and ST5 (5.6%) were comparatively rare. The presence of mixed STs in the same sample was also common (28.2%). Comparisons among children within the same household identified that shared ST profiles were common, but diversity within family units was also observed. Logistic regression analyses returned significant associations between the presence of Blastocystis, individual subtypes, or mixed subtypes for several variables. Intriguingly, the presence of animals was one of the most common significant associations. Taken together, these data represent an important step forward in understanding both the potential routes and risk factors that may influence Blastocystis transmission and will be useful in shaping future studies which seek to clarify the relationships between STs, pathogenicity, and zoonotic transmission.

Decreased fecal calprotectin levels in Spondyloarthritis patients colonized by Blastocystis spp.

Spondyloarthritis (SpA) is a group of chronic inflammatory systemic diseases mainly characterized by inflammation in the spine and/or peripheral joints. Although a link between SpA-pathogenesis, intestinal inflammation and gut dysbiosis has been proposed, studies have been focused on bacteria-host interactions and very little has been reported regarding intestinal parasites. Here, intestinal parasitic infection of 51 SpA-patients were evaluated and compared to healthy control individuals. No significant differences in the frequency of any parasite between SpA-patients and control individuals were found. Significantly higher levels of fecal calprotectin (FCP) were found in the SpA-patients compared to the control individuals. However, FCP levels were the same when comparing SpA-patients and control individuals, both colonized by Blastocystis spp. On the other hand, when comparing Blastocystis spp. colonized and Blastocystis spp. free SpA-patients, FCP levels were significantly higher in those Blastocystis spp. free. Without ignoring the small sample size as a study limitation, the results showed that in the SpA-patients colonized by Blastocystis spp., the FCP levels were significantly lower than those in the Blastocystis spp. free group and comparable to those in the control group. These findings seem to suggest a relationship between Blastocystis spp. and intestinal inflammation in SpA-patients, but studies intended to explore that interaction specifically should be designed.

Frequency and distribution of Blastocystis sp. subtypes in patients with spondyloarthritis in Bogotá, Colombia.

Although Blastocystis sp. is one of the most prevalent intestinal parasites worldwide, its role as a pathogen remains unclear. The use of molecular techniques to assess the genetic heterogeneity of Blastocystis sp. has become important to understand its function in some intestinal pathologies and if it is a key component of intestinal microbiota. Spondyloarthritis is a group of immune-mediated autoinflammatory diseases in which microbial dysbiosis in the gut (including parasites, bacteria and fungi) and intestinal inflammation are common features apparently associated with the pathophysiology of these disorders. This study included 74 patients diagnosed with spondyloarthritis and 57 systemically healthy individuals (included as controls), who were screened for intestinal parasites. Blastocystis sp. was detected in 68% and 73% of the patients with spondyloarthritis and controls, respectively. In faecal samples positive for Blastocystis sp., an 18S rRNA gene fragment of Blastocystis sp. was amplified and sequenced to identify their genetic sub-types. Patients with spondyloarthritis showed similar frequencies of ST1, ST2 and ST3 subtypes of Blastocystis sp. (30% each). The same subtypes were observed in controls, wherein almost 60% of the samples showed ST3. In addition, ST6 was found only in one sample from each group. ST1 subtype showed the greatest genetic variability. Although the same subtypes were detected in both patients with spondyloarthritis and controls, subtype prevalence studies conducted in Colombia indicate an association between ST3 and individuals with irritable bowel syndrome. This opens an interesting research avenue to further study of the epidemiology of Blastocystis sp. and its possible relationship with intestinal conditions in immunocompromised patients.

Multilocus genotyping of Giardia intestinalis in pet dogs of Medellín Colombia.

According to a few parasitological and epidemiological studies, Giardia is the most prevalent parasitic infection among pet dogs in the city of Medellín, the second-largest city in Colombia. This study determined the assemblages of Giardia in the fecal samples of dogs obtained from 18 veterinary centers of Medellín. One hundred fecal samples of dogs diagnosed with Giardia using microscopy were analyzed via nested polymerase chain reaction (PCR) using three genes (gdh, bg, and tpi). The PCR products were purified and sequenced, and phylogenetic analyses were conducted using the maximum likelihood algorithm of the three loci. From the 100 samples analyzed, 47 were Giardia-positive via PCR. Genotypes C and D were detected in six samples, neither of which were associated with human infection. However, the zoonotic potential of Giardia cannot be ruled out because of the small number of samples that could be sequenced for assemblage assignation.

Assembly and release of Dengue virus: Role of Alix protein / Ensamblaje y liberación del virus dengue: controversia sobre la participación de la proteína alix

RESUMEN Algunos virus envueltos usurpan la maquinaria celular ESCRT (complejo de clasificación endosomal requerido para el transporte) para llevar a cabo funciones como la transcripción, la traducción, el ensamblaje y la liberación de partículas virales desde las células huésped. Aunque esta estrategia ha sido estudiada principalmente en retrovirus, son varios los virus envueltos que la usan. El objetivo del trabajo fue explorar la participación de una proteína accesoria de ESCRT, la proteína Alix, en la transcripción, traducción, ensamblaje y liberación del virus dengue (DENV), así como su interacción con la proteína viral NS3. Células A549 infectadas con DENV2 fueron tratadas con pequeños ARN de interferencia (siRNA) para disminuir la expresión ("knock-down") de la proteína Alix. Simultáneamente, se obtuvo una línea A549 que expresaba una proteína NS3 recombinante y sobre este sistema se hicieron ensayos de inmunoprecipitación y "pull-down" para detectar interacción entre NS3 y Alix. Los resultados mostraron que el "knock-down" de Alix no tuvo efecto notable en la transcripción o la traducción viral, pero sí en el ensamblaje y la liberación de DENV2, mientras que los ensayos de "pull-down" revelaron la interacción entre NS3 y Alix. La participación de Alix en la producción de DENV2 y su interacción con NS3 constituyen un potencial blanco para el diseño de estrategias dirigidas a controlar la propagación de DENV.

ABSTRACT Since the finding that HIV recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to accomplish viral budding, this strategy has emerged as an escape route for enveloped viruses also. The work aimed to explore the participation of the cellular protein Alix (a human protein that acts as an adapter in the ESCRT pathway) on the transcription, protein expression, assembly and release of Dengue virus (DENV), and explore for its potential interaction with the viral protein NS3. To this purpose, A549 cells were infected with DENV2 and treated with small interfering RNAs (siRNA) to generate an Alix stable knockdown cells line. Also, an A549 cells line expressing a histidine-tagged NS3 protein was obtained. Both cells lines were used in immunoprecipitation and pull-down assays to assess the interaction between NS3 and Alix. The results showed that Alix knockdown had no effect on viral transcription or viral protein expression but influenced the assembly and release of DENV2 negatively. Finally, pull-down assays revealed the interaction between NS3 and Alix. The finding of an Alix participation in the production of DENV2 and its interaction with NS3 provides a potential target for the design of control/inhibition strategies against DENV spread.

Intestinal parasitic infections and associated factors in children of three rural schools in Colombia. A cross-sectional study.

Rural children are one of the populations that are most vulnerable to gastrointestinal parasite infections. Such diseases decrease the quality of life and result in growth and cognitive delays in the long term. This cross-sectional study was conducted to determine the frequency of intestinal parasite infections among rural schoolchildren in the municipality of Apulo, Colombia. A total of 97 stool samples from children aged between 5 and 15 years were collected and examined via direct light microscopy. Microscopic examination was repeated with sediments obtained using a fecal parasite concentrator, and the Kato-Katz test was performed. Frequency of intestinal parasite infection was 100%. Endolimax nana (77.35%), Blastocystis sp. (71.1%), Giardia intestinalis (39.1%), Entamoeba coli (25.7%), and the Entamoeba histolytica/dispar/moshkovskii complex (9.2%) were the most prevalent protozoa. Trichuris trichiura was the most prevalent helminth (12.3%), followed by Enterobius vermicularis (6.15%) and Ascaris lumbricoides (5.1%). Among the analyzed associated factors, consumption of untreated water increased the risk of acquiring pathogenic intestinal parasites. Finally, because G. intestinalis was the most prevalent pathogenic protozoan, molecular analysis was conducted to establish genetic assemblages and subassemblages of Giardia through sequence-based genotyping of the glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin genes. A total of 14 G. intestinalis-positive samples were genotyped, which revealed the presence of subassemblages AI (n = 1), AII (n = 7), BIII (n = 2), BIV (n = 2), and BIII/BIV (n = 1) as well as a mixed subassemblage AII + BIII (n = 1). Our results indicate that gastrointestinal parasite infections in the tested population were mainly caused by suboptimal water quality. Moreover, molecular typing of G. intestinalis suggested contamination of water by animal- and human-derived cysts.

Systematic Comparison of Strategies to Achieve Soluble Expression of Plasmodium falciparum Recombinant Proteins in E. coli.

Constructs containing partial coding sequences of myosin A, myosin B, and glideosome-associated protein (50 kDa) of Plasmodium falciparum were used to challenge several strategies designed in order to improve the production and solubility of recombinant proteins in Escherichia coli. Assays were carried out inducing expression in a late log phase culture, optimizing the inductor concentration, reducing the growth temperature for induced cultures, and supplementing additives in the lysis buffer. In addition, recombinant proteins were expressed as fusion proteins with three different tags (6His, GST, and MBP) in four different E. coli strains. We found that the only condition that consistently produced soluble proteins was the use of MBP as a fusion tag, which became a valuable tool for detecting the proteins used in this study and did not caused any interference in protein-protein interaction assays (Far Western Blot). Besides, we found that BL21-pG-KJE8 strain did not improve the solubility of any of the recombinant protein produced, while the BL21-CodonPlus(DE3)-RIL strain improved the expression of some of them independent of the rare codon content. Proteins with rare codons occurring at high frequencies (¼ 10%) were expressed efficiently in strains that do not supplement tRNAs for these triplets.

In vitro interaction between Plasmodium falciparum myosin B (PfMyoB) and myosin A tail interacting protein (MTIP).

Apicomplexan parasites, including Plasmodium falciparum, are obligate intracellular organisms that utilize a strategy termed "gliding" to move and invade host cells, causing disease. Gliding is carried out by a protein complex known as the glideosome, which includes an actin-myosin motor. To date, six myosins have been identified in P. falciparum (PfMyoA, B, C, D, E, and F), but only the role of PfMyoA, the myosin of the glideosome that is involved in the process of red blood cell and mosquito cell invasion, has been established. Based on previous observations, we speculated that PfMyoA and PfMyoB may have similar or redundant functions. To test this hypothesis, we searched for in vitro interactions between PfMyoB and MTIP (myosin A tail interacting protein), the myosin light chain of PfMyoA. A set of differentially tagged PfMyoA, PfMyoB, and MTIP recombinant proteins was employed to specifically and simultaneously detect each myosin in competition assays and inhibition assays using specific peptides. MTIP potentially acts as the light chain of PfMyoB.

[Polyclonal antibodies against recombinant dengue virus NS3 protein].

INTRODUCTION: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of themost attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. OBJECTIVE: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. MATERIALS AND METHODS: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. RESULTS: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling. CONCLUSION: The recombinant proteins expressed and the antibodies produced constitute valuable tools for studying DENV infectious processes involving NS3 and for evaluating tests designed to interfere with its functions.

Myosin B of Plasmodium falciparum (PfMyoB): in silico prediction of its three-dimensional structure and its possible interaction with MTIP.

The mobility and invasion strategy of Plasmodium falciparum is governed by a protein complex known as the glideosome, which contains an actin-myosin motor. It has been shown that myosin A of the parasite (PfMyoA) is the myosin of the glideosome, and the interaction of PfMyoA with myosin tail domain interacting protein (MTIP) determines its correct location and its ability to function in the complex. Because PfMyoA and myosin B of P. falciparum (PfMyoB) share high sequence identity, are both small proteins without a tail domain, belong to the class XIV myosins, and are expressed in late schizonts and merozoites, we suspect that these myosins may have similar or redundant functions. Therefore, this work examined the structural similarity between PfMyoA and PfMyoB and performed a molecular docking between PfMyoB and MTIP. Three-dimensional (3D) models obtained for PfMyoA and PfMyoB achieved high scores in the structural validation programs used, and their superimposition revealed high structural similarity, supporting the hypothesis of possible similar functions for these two proteins. The 3D interaction models obtained and energy values found suggested that interaction between PfMyoB and MTIP is possible. Given the apparent abundance of PfMyoA relative to PfMyoB in the parasite, we believe that the interaction between PfMyoB and MTIP would only be detectable in specific cellular environments because under normal circumstances, it would be masked by the interaction between PfMyoA and MTIP.

Anticuerpos policlonales contra la proteína recombinante NS3 del virus del dengue / Polyclonal antibodies against recombinant dengue virus NS3 protein

Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli / Producción de proteínas recombinantes de Plasmodium falciparum en Escherichia coli

Introduction: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum . However, the proteins of P . falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. Objective: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8). Materials and methods: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice. Results: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli , compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies. Conclusion: The use of genetically modified strains of E . coli was essential to achieve high expression levels of the four evaluated P . falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P . falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Introducción. La producción de proteínas recombinantes es fundamental para el estudio funcional de las proteínas de Plasmodium falciparum . Sin embargo, las proteínas recombinantes de P . falciparum están entre las más difíciles de expresar y, cuando lo hacen, usualmente se agregan dentro de cuerpos de inclusión insolubles. Objetivo. Evaluar la producción de cuatro proteínas de P. falciparum usando como sistema de expresión dos cepas de Escherichia coli genéticamente modificadas para favorecer la producción de proteínas heterólogas y establecer una reserva de proteínas recombinantes puras y solubles, y producir anticuerpos policlonales a partir de ellas. Materiales y métodos. Las proteínas recombinantes, las cuales correspondían a secuencias parciales de PfMyoA (Miosina-A) y PfGAP50 (proteína-asociada a glideosoma de 50 kDa) y a las secuencias completas de PfMTIP (proteína de interacción con miosina-A) y PfGAP45 (proteína asociada a glideosoma de 45 kDa), fueron expresadas como proteínas de fusión con la glutatión S-transferasa y luego purificadas y usadas para producir anticuerpos policlonales en ratón. Resultados. La expresión de las proteínas recombinantes fue mucho más eficiente en la cepa BL21-CodonPlus (la cual expresa tRNAs escasos en las bacterias silvestres), que en la cepa BL21-pG-KJE8. Por el contrario, aunque la cepa BL21-pG-KJE sobreexpresa chaperonas, no redujo la formación de cuerpos de inclusión. Conclusión. El uso de cepas de E . coli genéticamente modificadas fue fundamental para alcanzar altos niveles de expresión de las cuatro proteínas recombinantes evaluadas y permitió obtener dos de ellas en forma soluble. La estrategia utilizada permitió expresar cuatro proteínas recombinantes de P . falciparum en cantidad suficiente para inmunizar ratones y producir anticuerpos policlonales y, además, conservar proteína pura y soluble de dos de ellas para ensayos futuros.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

INTRODUCTION: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  OBJECTIVE: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  MATERIALS AND METHODS: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  RESULTS: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  CONCLUSION: The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Determinación del ensamblaje genético de aislados axénicos colombianos de Giardia intestinalis / Genetic assemblages of axenic Colombian cultures of Giardia intestinalis

Resumen Objetivo: Genotipificar muestras de Giardia aisladas de pacientes de tres regiones geográficas de Colombia. Materiales y métodos: Se examinaron 22 aislados de G. intestinalis, los primeros descritos cultivados axénicamente de origen colombiano por medio del análisis del polimorfismo de longitud de fragmentos de restricción (RFLP) del gen glutamato deshidrogenasa (gdh). Resultados y conclusión: El patrón del RFLP mostró que todos los aislados pertenecían al ensamblaje A, específicamente al AI; resultado confirmado por secuenciación. Con este estudio se pretende contribuir al conocimiento de los genotipos circulantes de Giardia intestinalis en Colombia.

Abstract Objective: To genotype samples of G. intestinalis isolated from patients of three Colombian regions. Materials and methods: 22 isolates of Giardia (all of them established as axenic cultures) were examined by analyzing restriction fragment length polymorphism (RFLP) of the glutamate dehydrogenase gene (gdh). Results and conclusion: The RFLP patterns showed that all of the Giardia isolates belonged to the assembly A, specifically to AI. This result was confirmed by sequencing. This study aims to contribute to the knowledge of circulating genotypes of Giardia intestinalis in Colombia.

Muerte celular programada en protozoarios: el caso de Giardia intestinalis / Programmed cell death in protozoa: Giardia intestinalis’s case

Giardia intestinalis es considerado uno de los eucariotas más antiguos y su poca complejidad representa una valiosa oportunidad para desentrañar los misterios de procesos vitales de eucariotas más complejos. Esta característica única de G. intestinalis y el hecho de que su genoma esté completamente secuenciado y disponible, y que todo su ciclo de vida puede ser reproducido in vitro, hacen de este parásito un modelo ideal para estudiar mecanismos celulares, entre ellos, la muerte celular programada. Desde el punto de vista morfológico y molecular, la apoptosis es uno de los tipos más complejos de muerte celular programada, la cual es un proceso normal durante el desarrollo celular, y tiene un papel esencial en el control de la proliferación celular y en la respuesta a retos inmunológicos o a daños celulares. Recientemente, se ha reportado que en protozoos, entre ellos Giardia, podría ocurrir un tipo de muerte celular programada similar a la apoptosis y los resultados de nuestros laboratorios apoyan esta hipótesis; sin embargo, no se han identificado hasta el momento las moléculas relacionadas con los procesos de apoptosis en estos parásitos. La presente revisión abarca una descripción de la morfología y estructura de las formas de vida de G. intestinalis, de su ciclo biológico, de la parasitosis que causa y de las estrategias quimioterapéuticas para su tratamiento. Asimismo, se hace un repaso de lo que hasta ahora se conoce sobre apoptosis en protozoarios, y específicamente en G. intestinalis, y se describen algunos resultados de nuestro grupo que apoyan la existencia de muerte celular programada en este parásito.

Giardia intestinalis is an early-branching eukaryote and its low complexity represents a valuable opportunity to unravel the mysteries of essential processes in more complex eukaryotes. In addition, the genome of G. intestinalis is completely sequenced and its entire life cycle can be reproduced in vitro. All these characteristics make of Giardia an ideal model for studying cellular mechanisms, such as programmed cell death. Apoptosis is one of the most complex types of programmed cell death and plays an essential role during cell development, cell proliferation and immune response. Recently it has been reported that in Giardia can take place events that resemble apoptosis and although our results support this hypothesis, molecules involved in this process have not yet been identified. This review includes a description of the morphology and structure of G. intestinalis, its life cycle, the disease that causes and the strategies for its treatment. In addition, we review what is known about apoptosis in protozoa, and specifically in G. intestinalis, and describe some results from our group supporting the existence of apoptosis-like programmed cell death in this parasite.

The rabies virus phosphoprotein synthesis and subcellular localization are modified by nerve growth factor.

Rabies virus P protein participates as a regulating factor in viral transcription and replication; recent studies found an antitranscriptional and antireplicative effect of nerve growth factor (NGF) and Neurotrophin-3 (NT-3) in infected neuron cultures. We investigated here the specific effect of the neurotrophins on P protein, evaluating its synthesis and subcellular distribution in adult mouse dorsal root ganglia neuron cultures infected and treated with NGF or NT-3. The results showed that NGF, but not NT-3, caused an increase in the quantity of P protein and an accumulation of protein in neuronal bodies, revealing changes in transport to the neuritic processes.

[Production and characterization of a polyclonal antibody against rabies virus phosphoprotein]. / Producción y caracterización de un anticuerpo policlonal dirigido contra la fosfoproteína del virus de la rabia.

INTRODUCTION: The expression of recombinant viral proteins has been a useful tool to study molecular biology and pathogenesis of virus infections. Because commercial specific antibodies to rabies virus phosphoprotein (P) are currently unavailable, these antibodies must be generated de novo in order to study the role of P protein during the infectious process. OBJECTIVE: A polyclonal antibody was produced and characterized for use against the phosphoprotein (P) of rabies virus. The antibody was raised in rabbits with a recombinant viral phosphoprotein (P) produced in Escherichia coli. MATERIALS AND METHODS: Gene P coding for the viral phosphoprotein (P) was amplified by RT-PCR and cloned into the expression vector PinPoint Xa-1 T. The recombinant protein P was expressed in Escherichia coli, purified by affinity chromatography and used to produce a polyclonal antibody anti-P. The antibody anti-P was purified and characterized by immunocytochemistry, immunofluorescence, fluorometric CELL-ELISA and Western blotting. RESULTS: The recombinant viral phosphoprotein was successfully expressed as a 50 kd biotinylated fusion protein which corresponds to the whole protein P of rabies virus. The polyclonal antibody raised against this recombinant protein P was able to detect with high specificity, protein P in cultures of sensorial neurons infected with rabies virus. CONCLUSIONS: The P protein obtained from heterologous expression in Escherichia coli became a specific antigen that was used to produce a polyclonal antibody capable of detecting native P protein in rabies virus infected cells.

[Genotyping of the Plasmodium falciparum msp1 (block 2) and dhfr (codon 108) genes in field samples collected in four endemic Colombian localities]. / Genotipificación de los genes msp1 (bloque 2) y dhfr (codón108) de Plasmodium falciparum en muestras de campo recolectadas en cuatro localidades endémicas de Colombia.

INTRODUCTION: Plasmodium falciparum is a highly polymorphic parasite, which allows it to evade the host's immune response, spread drug resistance and favours transmission. OBJECTIVES: To analyse the genetic diversity of P. falciparum populations in samples from four endemic localities in Colombia. MATERIALS AND METHODS: 123 blood samples were collected on filter paper from patients with non-complicated P. falciparum malaria during 2002 to 2004. The samples were genotyped using polymerase chain reaction with specific primers for the polymorphic region of block 2 of the msp1 gene and the 108 codon of the dhfr gene. RESULTS: In msp1 block 2, 95.9% (118/123; 95% CI: 90.8-98.7) of the samples harboured MAD20; 6.5% K1 (8/123; 95% CI: 2.8-12.4) and 2.4% RO33 (3/123; 95% CI: 0.5-6.9). For the dhfrgene the mutant allele N 108 was found in all the samples amplified, T 108 in 3.2% and the wild type S108 in 34.1%. Taking together all the results from both genes, 61.8% (76/123; 95% CI: 52.6-70.4) of the samples were simple infections and 38.2% (47/123; 95% CI: 29.6-47.4) were mixed infections. MAD20/N108-S108 (30.1%) was the most frequent combination among the latter. CONCLUSIONS: Simple infections, i.e, a single allelic type in each one of the genes studied, prevailed among the circulating parasite populations. In this study the genetic composition of P. falciparum parasite populations was very homogeneous.

Genotipificación de los genes msp1 (bloque 2) y dhfr (codón108)de Plasmodium falciparum en muestras de campo recolectadas en cuatro localidades endémicas de Colombia / Genotyping of the Plasmodium falciparum msp1 (block 2) and dhfr (codon 108) genes in field samples collected in four endemic Colombian localities

Introducción. Plasmodium falciparum es un parásito altamente polimórfico, lo cual le permite evadir la respuesta inmune del hospedero, diseminar la resistencia a medicamentos y favorecer la transmisión. Objetivos. Analizar la diversidad genética de las poblaciones de P. falciparum en muestras de cuatro zonas endémicas de malaria en Colombia. Materiales y métodos. Se incluyeron muestras de sangre recolectadas en papel de filtro de123 pacientes con malaria no complicada por P. falciparum durante los años 2002 a 2004; la genotipificación se realizó mediante reacción en cadena de la polimerasa con iniciadores específicos para los marcadores moleculares de la región polimórfica del bloque 2 del gen msp1 y del codón 108 de dhfr. Resultados. En el bloque 2 del gen msp1 se detectó MAD20 en 95,9 por ciento (118/123; IC 95 por ciento: 90,8 a 98,7), K1 en 6,5 por ciento (8/123; IC 95 por ciento: 2,8 a 12,4) y RO33 en 2,4 por ciento (3/123; IC 95 por ciento: 0,5 a 6,9) de las muestras. Para el gen dhfr, el alotipo mutante N108 se detectó en todas las muestras analizadas y el alotipo T108 en 3,2 por ciento (4/123; IC 95 por ciento: 0,9 a 8,1); el alotipo silvestre S108 se encontró en 34,1 por ciento (42/123; IC 95 por ciento: 25,8 a 43,2). Al combinar los resultados de ambos genes, el 61,8 por ciento (76/123; IC 95 por ciento: 52,6 a 70,4) de las muestras correspondieron a infecciones simples y el 38,2 por ciento (47/123; IC 95 por ciento: 29,6 a 47,4) a infecciones mixtas, siendo MAD20/N108-S108 la combinación más frecuente entre estas últimas (30,1 por ciento). Conclusiones. Las infecciones simples, o sea, la presencia de un solo alelo en cada uno de los genes, predominaron en las muestras estudiadas; las poblaciones de parásitos analizadas fueron muy homogéneas en su composición genética.