Multisystem Failure in Fatal Dengue: Associations between the Infectious Viral Serotype and Clinical and Histopathological Findings.

Dengue is the most important arthropod-borne viral infection of humans. However, its viral pathogenesis is still unknown. The information collected from dengue fatal cases is crucial for understanding the complex interactions between virulence and host factors. This study aimed to establish possible associations between the clinical characteristics, histopathological changes, replication, and tissue location of viral serotypes in dengue fatal cases. Clinical and histopathological characterizations, antigen localization in tissue, and detection of the infecting serotype and replication using real-time polymerase chain reaction were all performed on the dengue fatal cases. The majority of the cases involved people under the age of 20. Bleeding (48.3%), abdominal pain (44.8%), myalgia (52.9%), and headache (48.3%) were the most common clinical manifestations in the cases. There was multiorgan pathology, with histopathological changes primarily in the liver, spleen, and lung. Similarly, the viral antigen was found primarily in these organs; however, there were no associations between tissue changes, viral location, infecting serotypes, and replication processes. Dengue infection should be considered a multiorgan disease, the outcome of which is possibly not associated with the infecting viral serotype.

Genotyping of dengue virus from infected tissue samples embedded in paraffin.

Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history.

[Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2].

INTRODUCTION: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. OBJECTIVE: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. MATERIALS AND METHODS: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. RESULTS: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. CONCLUSIONS: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

Virus de la influenza / Influenza Virus

El virus de la influenza es un importante agente patógeno humano que causa infecciones respiratorias y una considerable morbimortalidad anual a nivel mundial. El virus puede circular esporádicamente durante brotes locales como parte de una epidemia estacional o puede generar una pandemia mundial. Durante las epidemias estacionales, la mortalidad se reporta principalmente entre personas muy jóvenes y adultos mayores; la Organización Mundial de la Salud estima que cada año se presentan entre tres y cinco millones de casos de enfermedad grave y de 250.000 a 500.000 muertes en el mundo. Las pandemias de influenza se presentan cuando se produce un reordenamiento genético del virus (antigen shift) que da lugar a una variante antigénicamente novedosa para la cual no hay anticuerpos en la población. Hasta la fecha se han reportado pandemias en 1918, 1957, 1968 y 2009, las cuales causaron la muerte de 60 millones de personas, aproximadamente.

[Detection of dengue virus antigen in post-mortem tissues]. / Detección de antígenos del virus del dengue en tejidos post mórtem.

The epidemiological situation of dengue has worsened over the last decade. The difficulties in preventing its transmission and the absence of a vaccine or specific treatment have made dengue a serious risk to public health, health centers and research systems at different levels. Currently, most studies on the pathogenesis of dengue infection focus on the T-cell immune response almost exclusively in secondary infections and are aimed at identifying the mechanisms involved in the development of vascular permeability and bleeding events that accompany the infection. This report describes the case of a baby girl less than 45 days of age with clinical signs of severe dengue, whose diagnosis was confirmed by reverse transcription polymerase chain reaction in post-mortem tissue samples and by the ancillary diagnostic use of immunohistochemistry, which detected viral antigens in all organs obtained at autopsy. This case highlights the importance of studying primary infections associated with severe dengue, particularly in children, who are more likely to develop the severe form of the disease without previous infection, and it further stresses the importance of a diagnosis that should not be based solely on the examination of liver tissue samples when studying the pathogenesis of the viral infection.

Detección de antígenos del virus del dengue en tejidos post mórtem / Detection of dengue virus antigen in post-mortem tissues

El panorama epidemiológico del dengue ha empeorado durante la última década. Las dificultades para prevenir su transmisión, así como la ausencia de una vacuna o tratamiento específico, lo convierten en un riesgo que desafía las medidas de salud pública y desborda la capacidad de los centros de salud y los sistemas de investigación a muchos niveles. Actualmente, la mayoría de los estudios sobre la patogenia de la infección centran su atención en la respuesta inmunitaria de las células T casi exclusivamente en infecciones secundarias y están dirigidos a identificar los mecanismos implicados en el desarrollo de la permeabilidad vascular y de los eventos hemorrágicos que lo acompañan. En este reporte se describe el caso de una menor de 45 días de edad con signos clínicos de dengue grave, cuyo diagnóstico se confirmó por reacción en cadena de la polimerasa de transcripción inversa en muestras de tejido post mórtem y por herramientas de apoyo diagnóstico de inmunohistoquímica, las cuales detectaron antígenos virales en todos los órganos obtenidos en la necropsia. Este caso subraya la importancia del estudio de las infecciones primarias asociadas a dengue grave, particularmente en niños, en quienes es más probable el desarrollo de la forma grave de la enfermedad sin una infección previa, y, además, pone de relieve la importancia de un diagnóstico que no se limite a las muestras de tejido hepático en el estudio de la patogenia de la infección viral.

The epidemiological situation of dengue has worsened over the last decade. The difficulties in preventing its transmission and the absence of a vaccine or specific treatment have made dengue a serious risk to public health, health centers and research systems at different levels. Currently, most studies on the pathogenesis of dengue infection focus on the T-cell immune response almost exclusively in secondary infections and are aimed at identifying the mechanisms involved in the development of vascular permeability and bleeding events that accompany the infection. This report describes the case of a baby girl less than 45 days of age with clinical signs of severe dengue, whose diagnosis was confirmed by reverse transcription polymerase chain reaction in post-mortem tissue samples and by the ancillary diagnostic use of immunohistochemistry, which detected viral antigens in all organs obtained at autopsy. This case highlights the importance of studying primary infections associated with severe dengue, particularly in children, who are more likely to develop the severe form of the disease without previous infection, and it further stresses the importance of a diagnosis that should not be based solely on the examination of liver tissue samples when studying the pathogenesis of the viral infection.

Glucógeno hepático en dengue severo: análisis histopatológico / Hepatic glucogen in cases with severe dengue: histopathological changes

Antecedentes: El virus del dengue afecta distintos órganos, pero se ha determinado que el hígado es el principal blanco de acción y en donde ocurre la mayor severidad del daño. Existen pocos estudios sobre los cambios histológicos durante la infección por dengue. Objetivos: Analizar las alteraciones histopatológicas post-mortem en hígados de pacientes que presentaron la forma grave del dengue. Métodos: Se revisaron los cortes de hígado de 20 pacientes con dengue severo y se realizaron coloraciones y pruebas para glucógeno. Resultados: Encontramos pérdida de glucógeno citoplasmático en todos los casos analizados y la presencia de glucógeno intranuclear en dos de ellos. Conclusiones: En este estudio se reporta por primera vez la presencia de masas de glucógeno intranuclear en hepatocitos de dos niños fallecidos con dengue grave.

Background: Dengue virus affects various organs, but the liver is the main target of damage and where the most severe damage can occur. There are few studies on the histological changes in the liver during dengue infection. Aims: To analyze the histopathological post-mortem alterations in livers from patients with Methods: We revised serial liver sections, which were stained and tested for glycogen, from 20 patients with severe dengue. Results: We found loss of cytoplasmic glycogen in all cases analyzed and the presence of intranuclear glycogen in two of them. Conclusions: This is the first report of the presence of intranuclear glycogen masses during severe dengue.

Alteraciones morfológicas en pulmón por la influenza A H1N1/v09 en autopsias, Colombia, 2009 / Morphological changes in lung tissue of victims associated with the 2009 A H1N1/v09 influenza pandemic in Colombia

Introducción. La influenza es una infección respiratoria aguda que se presenta de forma estacional y pandémica. En el 2009, la Organización Mundial de Salud (OMS) declaró una pandemia por influenza de tipo A en la que se reportaron en Colombia 3.876 casos de infección, de los cuales 239 fallecieron. Objetivo. Describir los cambios morfológicos asociados a la infección por el virus A H1N1/v09 en tejido pulmonar de autopsias de la pandemia de 2009 en Colombia.Materiales y métodos. Se estudiaron 75 casos con diagnóstico por RT-PCR para el virus A H1N1/v09, de los cuales, 20 fueron seleccionados para el estudio morfológico mediante microscopía convencional de luz, microscopía óptica de alta resolución y electrónica de transmisión e inmunohistoquímica.Resultados. De los 75 casos estudiados, 83 % presentaron pneumonitis viral y 17 % alveolitis. Se observaron complicaciones por hemorragia intraalveolar (66 %) y edema (89 %), daño alveolar difuso (2 %) e infección bacteriana concomitante (32 %).Algunos de los cambios morfológicos observados fueron: destrucción del epitelio alveolary el instersticio, edema, macrófagos con citoplasma vacuolado e infiltración de leucocitos polimorfonucleares en la luz alveolar y el intersticio; vacuolización citoplásmica en neumocitos de tipo I y cuerpos electrodensos en restos celulares en la luz alveolar; inmunorreacción de antígenos virales en el epitelio bronquiolar y en células del infiltrado alveolar. Conclusión. El porcentaje bajo de infección bacteriana concomitante observado en los casos de influenza A H1N1/ v09 en este estudio, es una característica sobresaliente que sugiere que el resultado fatal de la infección, probablemente no esté asociado a una enfermedad bacteriana secundaria, como se ha sugerido en reportes previos. Es probable que las lesiones observadas se puedan atribuir al daño tisular en la respuesta inflamatoria celular y humoral asociada a la infiltración por células poliformonucleares y macrófagos en el intersticio y la luz alveolares, como también por la lesión viral.

Introduction. Influenza is an acute respiratory infection that may be seasonal or pandemic. In 2009 The World Health Organization (WHO) declared an influenza pandemia; 3,876 cases and 239 deaths were reported in Colombia. Objective. The morphological changes in lung tissues associated with virus infection H1N1/v09 were described from autopsied victims.Materials and methods. Seventy-five cases were diagnosed by RT-PCR for influenza A H1N1/v09, of which the lungs of 20 were selected for morphological study by light microscopy, optical microscopy, high-resolution transmission electron microscopy and immunohistochemistry. Results. Of the 75 cases, 83% had viral pneumonitis and 17% alveolitis. Complications included intra-alveolar hemorrhage (66%), edema (89%), diffuse alveolar damage (2%), and bacterial co-infection (32%). Morphological changes were as follows: destruction of the alveolar epithelium and interstitium, edema, macrophages with vacuolated cytoplasm,and infiltration of polymorphonuclear leukocytes in the alveolar lumen and interstitium, vacuolization cytoplasmic type I pneumocytes and electron-dense bodies in cellular debris in the alveolar lumen, and immunoreactivity of viral antigens in bronchiolar epithelial cells and alveolar infiltrate. Conclusion. The low percentage of bacterial co-infection observed in these cases was a prominent feature, and suggested that the fatal result was probably not associated with secondary bacterial disease (Indicated by previous reports). The tissue lesions were attributed to tissue damage due to viral lesion, as well as the cellular and humoral inflammatory response associated with infiltration by polymorphonucleocytes and macrophages in the interstitium and alveolar lumen.

Effect of an Oxadiazoline and a Lignan on Mycolic Acid Biosynthesis and Ultrastructural Changes of Mycobacterium tuberculosis.

Tuberculosis (TB) is an important disease that causes thousands of deaths around the world. Resistance against antitubercular available drugs has been reported; so, research on new effective antimycobacterial molecules is needed. Antimycobacterial activity of three lignans and two synthetic hydrazones was assessed against Mycobacterium tuberculosis H37Rv by antimycobacterial microdilution assay (TEMA). An oxadiazoline (AC451) and a lignan (ethoxycubebin) were the most active compounds (MIC 6.09 and 62.4 µM, resp.). Several changes in mycolic acid profile of treated bacteria were detected with both compounds by mass spectrometry analysis. Additionally, the level of reduction of mycolic acids in ethoxycubebin treatment was correlated to disruption in bacterial morphology.

[Morphological changes in lung tissue of victims associated with the 2009 A H1N1/v09 influenza pandemic in Colombia]. / Alteraciones morfológicas en pulmón por la influenza A H1N1/v09 en autopsias, Colombia, 2009.

INTRODUCTION: Influenza is an acute respiratory infection that may be seasonal or pandemic. In 2009 The World Health Organization (WHO) declared an influenza pandemia; 3,876 cases and 239 deaths were reported in Colombia. OBJECTIVE: The morphological changes in lung tissues associated with virus infection H1N1/v09 were described from autopsied victims. Materials and methods. Seventy-five cases were diagnosed by RT-PCR for influenza A H1N1/v09, of which the lungs of 20 were selected for morphological study by light microscopy, optical microscopy, high-resolution transmission electron microscopy and immunohistochemistry. RESULTS: Of the 75 cases, 83% had viral pneumonitis and 17% alveolitis. Complications included intra-alveolar hemorrhage (66%), edema (89%), diffuse alveolar damage (2%), and bacterial co-infection (32%). Morphological changes were as follows: destruction of the alveolar epithelium and interstitium, edema, macrophages with vacuolated cytoplasm,and infiltration of polymorphonuclear leukocytes in the alveolar lumen and interstitium, vacuolization cytoplasmic type I pneumocytes and electronedense bodies in cellular debris in the alveolar lumen, and immunoreactivity of viral antigens in bronchiolar epithelial cells and alveolar infiltrate. CONCLUSION: The low percentage of bacterial co-infection observed in these cases was a prominent feature, and suggested that the fatal result was probably not associated with secondary bacterial disease (Indicated by previous reports). The tissue lesions were attributed to tissue damage due to viral lesion, as well as the cellular and humoral inflammatory response associated with infiltration by polymorphonucleocytes and macrophages in the interstitium and alveolar lumen.

Cuantificación de los melanocitos en la piel facial: otra ayuda para evaluar los bordes de resección del melanoma / Quantification of melanocytes in facial skin: another help for evaluation of resection borders of melanoma

Introducción: Los melanocitos epidérmicos están ampliamente separados entre sí, rodeados por un halo; son de citoplasma claro y núcleo picnótico, más pequeño que el de los queratocitos. En la cara es difícil diferenciar entre los cambios por exposición solar y un melanoma in situ, así como establecer si los bordes de resección de un melanoma in situ tienen tumor o si los melanocitos presentes sólo tienen cambios por el sol. Objetivo: Cuantificar el número de melanocitos en adultos normales y en los bordes de resección sin tumor, de carcinomas basocelulares y de melanomas in situ de la piel malar. Materiales y métodos: Se estudiaron veinticinco especímenes de piel tipo I-II de la mejilla de adultos mayores de 40 años, siete de autopsias de hombres, once de los bordes de carcinomas basocelulares y siete de los bordes de resección de melanomas in situ, libres de tumor. Con la coloración de hematoxilina-eosina, tres observadores contaron los melanocitos basales por milímetro lineal en cada espécimen, usando un fotomicroscopio Axiophot Zeiss. Resultados: En un milímetro lineal (3 campos de 40X), el número de melanocitos fue de 18±3 en la piel normal, de 22±7 en los bordes del carcinoma basocelular y de 30±9 en los del melanoma in situ. Conclusiones: El número máximo de melanocitos en un campo de 40X en los tejidos estudiados no debe exceder de 7,5±4 (30 melanocitos) por mm lineal. Un número mayor es una alerta que debe unirse a otros cambios para determinar si hay persistencia de melanoma in situ.

COMPARACIÓN HISTOLÓGICA Y MORFOMÉTRICA ENTRE EL OJO DE Eremophilus mutisii (TRICHOMYCTERIDAE) Y EL DE Oncorhynchus mykiss (SALMONIDAE) / Histological And Morphometrical Comparison Between Eremophilus mutisii (Trichomycteridae) And Oncorhynchus mykiss (Salmonidae) Eyes

La visión es de importancia variable dependiendo de la relación de los peces con su hábitat, siendo clave en algunas especies y secundaria en otras. El objetivo de este estudio fue realizar una comparación entre el ojo de capitán de la sabana y trucha arco iris. La primera, nativa del altiplano cundiboyacence. La segunda es una especie introducida al país y con alta importancia en la piscicultura. Se obtuvieron cortes de ojo a 5 µm de espesor en ambas especies. Las dos presentan la estructura ocular típica de teleósteos conformada por una retina con ocho capas y dos membranas, células fotorreceptoras (conos y bastones), así como los otros tipos de células neuronales características (bipolares, horizontales, amacrinas y ganglionares), y un cristalino casi esférico. La trucha presenta cartílago en la esclerótica y en capitán de la sabana está ausente. El espesor promedio de las estructuras en capitán de la sabana es: retina de 183,5 41,2 µm, córnea de 20,6 5,4 µm e iris de 31,2 6,4 µm; trucha arco iris presentó una retina de 389,5 65,2 µm, una córnea de 300,4 101,8 µm y un iris de 41,2 13,7 µm. Los resultados obtenidos en este estudio muestran diferencias en el espesor de la retina, cornea, esclerótica, tamaño relativo del ojo y diámetro horizontal del cristalino; para los cuales trucha arco iris presenta un valor mayor. Estas diferencias posiblemente se relacionan con diferencias en sensibilidad y resolución visual entre las dos especies y podrían reflejar adaptaciones del sistema visual al medio.

The importance of vision in fishes varies depending on their relationship to their habitat, being crucial for some species, and secondary for other species. The main goal of this work was to make a comparison between the eyes of capitán de la sabana and rainbow trout. The first one, from the cundiboyacense highlands, and the second one, a foreign species introduced in our country, very important as a cultured organism. Histological 5 µm thickness eye sections were obtained. Both species do present the typical teleosteal eye structure with an 8 layers retina, photoreceptor cells (cones and rods), as well as different types of neurons (bipolar, horizontal, amacrine, and ganglionic cells ), and an almost spherical lens. Rainbow trout has cartilage in the sclerotica, while capitán de la sabana does not present this tissue. Mean thickness of the structures in capitán de la sabana are: retina 183.5 41.2 µm, cornea 20.6 5.4 µm and iris 31.2 6.4 µm; mean thickness in rainbow trout are: retina 389.5 65.2 µm, cornea 300.4 101.8 µm, and iris 41.2 13.7 µm. Our results shows thickness differences in retina, cornea, sclerotica, relative eye size, lenses diameter, being larger for rainbow trout. These differences are probably related to differences in sensibility and visual resolution between the species, and do reflect visual system adaptations to different environments.

Cellular location of KMP-11 protein in Trypanosoma rangeli.

We describe the localization of the KMP-11 protein in the Trypanosoma rangeli parasite determined by immunoelectron microscopy using a monoclonal antibody generated against the Trypanosoma cruzi KMP-11 protein. The data reported herein show that the T. rangeli KMP-11 protein is mainly accumulated in the parasite cytoplasm, the coat, the flagellum, and the flagellar pocket. The high degree of sequence homology between the KMP-11 proteins from both parasites suggests that the KMP-11 protein from T. rangeli, like that of T. cruzi, could also be associated with the parasite cytoskeleton.

[Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae)]. / Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae).

Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04 +/- 4.00 microm in length and 13.96 3.70 microm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08% of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90%) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week.

[Image analysis to quantify S100-positive dendritic cells in leprosy-affected skin]. / Utilización de análisis de imágenes para cuantificar células dendríticas S100 positivas en piel con lepra.

A morphometric analysis of skin dendritic cells was done on biopsies of patients with different forms of leprosy. An anti S100 antibody was used to determine dendritic cell quantity and extension. Patients with a better immune response to the bacillus showed a greater number of dendritic cells in the cases of dimorphic tuberculoid leprosy and tuberculoid leprosy. This result contrasted with that from patients with dimorphic lepromatous leprosy and lepromatous leprosy.

Utilización de análisis de imágenes para cuantificar células dendríticas S100 positivas en piel con lepra / Image analysis to cuantify S100 - positive dendritic cells in leprosy - affected skin

Se realizó un análisis morfométrico de células dendríticas de piel, que incluyó la determinación de la cantidad y el área ocupada, empleando un anticuerpo anti-S100 en biopsias de pacientes con diferentes tipos de lepra. El análisis reveló las diferencias entre los pacientes que presentaban una mejor respuesta inmune al bacilo, reflejada en una mayor cantidad de células dendriticas en los casos de lepra dimorfa tuberculoide y lepra tuberculoide con respecto a aquellos pacientes con lepra dimorfa lepromatosa y lepra lepromatosa.

[In vitro encystation of Giardia lamblia: analysis with two-dimensional electrophoresis of differentially expressed proteins]. / Enquistación in vitro de Giardia lamblia: análisis por electroforesis bidimensional de proteínas expresadas diferencialmente.

The reconstruction of Giardia lamblia life cycle in vitro is an excellent tool for the study of the parasite's molecular biology. The present work describes techniques developed that better define parasite differentiation. An encystation protocol is presented along with a method for isolation and purification of the produced cysts. The cyst morphology at the light microscopy level is identical to that of in vivo cysts. A two-dimension protein map obtained by high-resolution electrophoresis indicated that most of the parasite's proteins are acid. Based on this result, the two dimension gel electrophoresis used a pH 4-7 gradient in the first, isoelectric focusing dimension. Differences in protein expression during the stages of encystation were clearly discerned, as well as images of the parasite obtained by light and by transmission electron microscopy that describe the morphological and the ultrastructural changes that occur as the cysts are produced in vitro.