BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Abstract INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication.

Objetivo: Evaluar, en condiciones in vitro , la actividad antibacterial de los extractos de cuerpos grasos y de la hemolinfa de larvas de tercer estadio de Sarconesiopsis magellanica , la cual se comparó con los efectos obtenidos de las mismas sustancias derivadas de Lucilia sericata . S. magellanica (Diptera: Calliphoridae) es una mosca de importancia principalmente forense, utilizada en la determinación del intervalo post mortem . Por sus hábitos necrófagos, es considerada un modelo potencialmente útil en terapia larval. Material y métodos: Se extrajeron los cuerpos grasos de las larvas mediante la técnica de disección corporal y la hemolinfa se obtuvo mediante decapitación y centrifugación de los especímenes larvales. Las bacterias evaluadas fueron Staphylococcus aureus y Pseudomonas aeruginosa . Los métodos utilizados para evaluar la actividad antibacterial fueron difusión en agar y unidades formadoras de colonias (UFC/ml). Resultados: Después de la correspondiente incubación, los resultados generales mostraron que la actividad antibacterial de la hemolinfa y de los cuerpos grasos, tanto de L. sericata como de S. magellanica , fueron efectivos contra S. aureus y P. aeruginosa sin diferencias significativas entre las especies de moscas, aunque con algunas diferencias entre las cepas bacterianas. Conclusiones: Los resultados obtenidos sugieren que estas sustancias podrían tener un efecto similar en el tratamiento de heridas infectadas contra los microorganismos evaluados.

Objective: This study aimed to evaluate the in vitro antibacterial activity of fat body and hemolymph extracts from Sarconesiopsis magellanica (Diptera: Calliphoridae) third-instar larvae, compared to the effect obtained using the same extracts but derived from Lucilia sericata . S. magellanica blowflies are considered important in forensic sciences due to their usefulness in determining the post mortem interval. This blowfly could be useful in larval therapy due to its necrophagous habits. Materials and methods: Fat body from larvae was removed by dissection, and hemolymph via decapitation and centrifugation of larval specimens. The antibacterial effect was tested against Staphylococcus aureus and Pseudomonas aeruginosa using two methods: agar diffusion and colony forming units (CFU/mL). Results: Hemolymph and fat body extracts derived from both L. sericata and S. magellanica were effective against S. aureus and P. aeruginosa , with no significant differences between blowfly species, although with some differences between the bacterial strains. Conclusions: The results obtained suggest that S. magellanica and L. sericata fat body and hemolymph extracts might have a similar antimicrobial activity against these microorganisms when used to treat infected wounds.

La primera línea celular de Aedes aegypti fue establecida por Grace en 1966 y desde entonces se han utilizado para el estudio de virus, bacterias y parásitos. En el presente trabajo se describen, por primera vez, algunas características citoquímicas de los cultivos celulares de A. aegypti, infectados con la cepa (MHOM/CO/87CL412) de Leishmania panamensis. También se realizó un estudio morfológico de las células del cultivo. Se observaron 30 células pequeñas con apariencia fibrolastoide de 10.84±2.54 µm de largo y 5.31±1.26 µm de ancho; otras 30 presentaron apariencia epitelioide con 23.04±4.00 µm de largo y 13.96±3.70 µm de ancho; éstas últimas predominaron sobre las de apariencia fibroblastoide. De 113 células, un 7.08%, presentaron abundantes gránulos citoplasmáticos positivos con la coloración de PAS, indicando presencia de polisacáridos. La prueba de peroxidasa dio un resultado negativo. El mayor porcentaje de infección (18.90%), de un total de 101 células, se presentó el día 6. Ultraestructuralmente, las células presentaron un citoplasma con aspecto vacuolado; algunas contenían parásitos, otras material fibrilar y otras estaban vacías. Los resultados indican que los cultivos celulares de A. aegypti pueden ser infectados por L. panamensis y mantener dicho proceso por aproximadamente una semana.

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 ºC, 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 µm 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 µm in length and 5.31±1.26 µm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04±4,00 µm in length and 13.96±3.70 µm 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. Rev. Biol. Trop. 56 (2): 447-458. Epub 2008 June 30.

Introducción. Los cultivos celulares de insectos son una metodología útil en estudios biomédicos y tecnológicos. Objetivo. El propósito principal del presente trabajo fue obtener y caracterizar cultivos celulares derivados de tejidos embrionarios de Aedes aegypti. Materiales y métodos. Se emplearon huevos embrionados para los explantes de tejidos en los medios de cultivos MM/VP12 y L-15/Grace, con suplemento de 20% de suero fetal bovino y una mezcla al 1% de antibiótico y antimicótico, con un rango de pH entre 6,8 y 7,0. Los cultivos se incubaron a una temperatura de 28ºC sin atmósfera de CO2. Resultados. El crecimiento celular se obtuvo en el medio L-15/Grace, 3 semanas después de haber sido sembrados los tejidos embrionarios; sin embargo, se necesitaron 6 meses para la formación de la monocapa confluente. Desde agosto de 2003 hasta junio de 2004, se habían realizado 28 subcultivos. Las células se caracterizaron morfológicamente; predominaron las formas epitelioides en subcultivos de pases altos. También se reconocieron las particularidades morfométricas del cariotipo y, además, se determinaron los perfiles isoenzimáticos y moleculares de los cultivos celulares, los cuales se compararon con muestras de adultos de la especie tomadas de la misma colonia y con líneas celulares derivadas de otros insectos. Discusión. Estas células representan, potencialmente, un importante sistema in vitro en investigaciones básicas y aplicadas.

Introduction. Cell cultures from insects are a useful methodology in technological and biomedical studies. Objective. The present work was aimed at obtaining and characterizing cell cultures derived from Aedes aegypti embryonic tissues. Materials and methods. Embryonated eggs were used for embryonic tissue explants in L-15/Grace and MM/VP12 culture media, supplemented with 20% fetal bovine serum and a mixture of 1% antimycotic and antibiotics, at a pH ranging from 6.8 to 7.0. The incubation temperature was 28º C; a CO2 atmosphere was not required. Results. Cell growth was obtained in L-15/Grace medium three weeks after embryonic tissues explants. Six months were required for achieving a confluent monolayer. Twenty eight serial cell subcultures were carried out from August 2003 to June 2004. Cell morphology was characterized as being epithelial in subcultures of high passages; karyotype morphometric characteristics were recognized and the molecular and isozymatic profiles were also established. The cultures were compared with adult samples from the species taken from the same colony and with cell lines derived from other insects. Discussion. These cells are an important in vitro system in applied and basic research.

Lutzomyia spinicrassa is a vector of Leishmania braziliensis in Colombia. This sand fly has a broad geographical distribution in Colombia and Venezuela and it is found mainly in coffee plantations. Baseline biological growth data of L. spinicrassa were obtained under experimental laboratory conditions. The development time from egg to adult ranged from 59 to 121 days, with 12.74 weeks in average. Based on cohorts of 100 females, horizontal life table was constructed. The following predictive parameters were obtained: net rate of reproduction (8.4 females per cohort female), generation time (12.74 weeks), intrinsic rate of population increase (0.17), and finite rate of population increment (1.18). The reproductive value for each class age of the cohort females was calculated. Vertical life tables were elaborated and mortality was described for the generation obtained of the field cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.

Con el propósito de establecer la prevalencia de leishmaniosis canina se efectuó un estudio descriptivo en 307 caninos, ubicados en 17 veredas de los municipios de Neiva, Tello y Algeciras, a los cuales se les practicó examen clínico, punción y aspiración del ganglio linfático poplíteo para extendido y coloración en lámina; toma de sangre completa por venopunción para análisis mediante la técnica de inmunofluorescencia indirecta. La población canina estuvo conformada por perros mestizos con edad promedio de tres años; el 30,6 % fueron hembras y el 69,4 % machos. En el examen físico de los caninos se observó: enflaquecimiento, 31 %; onicogrifosis, 29,3 %; linfadenitis de ganglio poplíteo 3,2 %; áreas alopécicas 21,8 %; lesiones eritematosas 11,5 % y úlceras cutáneas 3,3 %. El 1,4 % de los caninos analizados presentaron amastigotes en el aspirado y el 17,2 % fueron seropositivos. La prevalencia de anticuerpos contra L. chagasi es alta comparada con otros estudios realizados en Colombia y otros países, corroborando la endemicidad de la enfermedad en el área. Por consiguiente, es necesario fortalecer las estrategias de vigilancia epidemiológica y la búsqueda activa de casos humanos.

With the purpose of establishing the prevalence of canine leishmaniasis a descriptive study was carried out in 17 villages of the municipalities of Neiva, Tello and Algeciras, in the department of Huila, Colombia. The selected canine population was composed of 307 dogs, which were submitted to clinical examination, stained smears of lymph node needle aspiration, and bleeding for serological analysis by the Immunofluorescent Antibody Tests (IFAT). The canine population was conformed by mestizo dogs, with a 3 year age average; 30,6 % were females. Upon physical examination loss of weight was observed in 31% of dogs; lynfadenopathy of the popliteal node in 3,2 %; alopecia in 21,8 %, eritematous lesions in 11,5 % and skin ulcers in 3,3 %. 17,2 % of the dogs has a positive serology and in 1.43% of them, amastigotes were observed in the smears of lymph node aspirates. These prevalence of anti-L. chagasi antibodies is relatively high compared with other studies in Colombia and in other countries. The present study, confirms the endemic nature of the illness in the area, high lighting the need for strengthening public health surveillance and active search for human cases.

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.