Apoptosis in HEp-2 cells infected with Ureaplasma diversum

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location ofUreaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversuminvasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

Dos aislamientos de Salmonella Infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular

En este trabajo se investigó la presencia de determinantes característicos de plásmidos de virulencia en dos aislamientos clínicos de Salmonella Infantis portadores de plásmidos de multirresistencia. Además, se estudió la capacidad de invasión y proliferación en células eucariotas no fagocíticas. Ninguno de los aislamientos de S. Infantis mostró los determinantes genéticos que caracterizan a los plásmidos de virulencia para este género (operón spv). Los ensayos de invasión sobre líneas celulares eucariotas mostraron que los aislamientos de S. Infantis presentan una capacidad de invasión disminuida pero persisten y proliferan en el citoplasma, independientemente de utilizar una línea celular permisiva (HeLa) o no permisiva (NRK) para tal fin. Finalmente, no se observaron indicios microscópicos que podrían hacer sospechar un efecto bactericida de estas líneas celulares sobre los aislamientos estudiados.

Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spv operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to Invade but they remained and proliferated In the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the Isolates tested.

Apoptotic effect on HeLa cells produced by Chlamydia trachomatis-LPS

La interacción entre el lipopolisacárido (LPS) de Chlamydia trachomatis y las células de mamíferos permanece sin ser dilucidado. Chlamydia trachomatis es una bacteria intracelular responsable de diversas enfermedades en los humanos y animales. En este trabajo mediante el aislamiento del lipopolisacárido de dos serovares de Chlamydia trachomatis (LGV1-LGV2) y usando una coloración Supravital fluorescente (Hoechst 33258) fue posible investigar la respuesta de las células HeLa. El efecto apoptótico que sufren este tipo de células fue visible cuando fueron expuestas a dicho LPS en concentraciones iguales o mayores que 0,5 µg/mL por un periodo de 48 horas, sin embargo se observó la falta de repuesta celular en su ausencia o en presencia de LPS de otras bacterias. Adicionalmente, el uso en iguales condiciones de polimyxina B conocido como un neutralizador de la acción del LPS demostró una disminución del efecto apoptótico en dichas células, indicando que la respuesta celular observada fue producida por C. trachomatis-LPS. Los resultados de este trabajo le dan fuerza a la teoría de que el LPS de C. trachomatis pudiera ser el responsable del efecto tóxico que se observa sobre las células cervicales infectadas con esta bacteria intracelular.

Adherence to HeLa cells, typing by killer toxins and susceptibility to antifungal agents of Candida dubliniensis strains

The aim of this study was to evaluate the adherence capability to HeLa cells, the susceptibility to killer toxins and the in vitro susceptibility to antifungal agents (eTest? method - AB Biodisk, Solna, Sweden) of 9 Candida dubliniensis isolates recovered from HIV+ and AIDS patients. The adherence test was strongly positive for strain ATCC 777 and positive for all other strains. Typing by killer toxins revealed two different biotypes among the 9 isolates studied: 888 and 688. Only biotype 688 (ATCC 777) was susceptible to the K2 toxin. There was a significant inverse correlation between adherence and killer toxin susceptibility (r = -0.8525 - p = 0.0035). No strains presented resistance to fluconazole, itraconazole, ketoconazole, voriconazole, flucytosine or amphotericin-B. With the exception of ATCC 777, all the other isolates presented similar behavior.

O objetivo do presente trabalho foi avaliar o comportamento de cepas de Candida dubliniensis recuperadas de pacientes HIV+ e com AIDS por meio da pesquisa de capacidade de adesão a células HeLa, susceptibilidade a toxinas "Killer" e resistência in vitro a antifúngicos (eTest® AB Biodisk, Solna, Suécia). O ensaio de adesão foi fortemente aderente para a amostra padrão ATCC 777, e aderente para os demais isolados. Os testes de tipagem das amostras frente às cepas-padrão produtoras de toxinas "Killer" mostraram dois biótipos diferentes dos 9 isolados estudados: 888 e 688. Somente o biótipo 688 (ATCC 777) de C. dubliniensis foi sensível à toxina K2. Houve correlação inversa significativa entre adesão e sensibilidade a toxinas "killer" (r = -0,8525 - p = 0,0035). Em relação à pesquisa de resistência a antifúngicos, as amostras de C. dubliniensis foram sensíveis ao fluconazol, itraconazol, cetoconazol, voriconazol, à flucitosina e anfotericina B. Com exceção da amostra ATCC 777, todas as demais mostraram comportamento similar.

Sorotipos de Escherichia coli enteropatogênica clássica (EPEC) causadores de alteraçöes da ultra-estrutura do intestino delgado e invasäo de células Hela / Serotypes of classical enteropathogic that cause changes in the small bowel ultrastructure and invasion of Hela cells

Escherichia coli enteropatogênica clássica (EPEC) é a principal causa de diarréia em lactentes menores de um ano de idade nos países subdesenvolvidos. In vitro, cepas de EPEC sao capazes de aderir a células HeLa ou Hep-2, obedecendo um padrao característico denominado adesao localizada. Estudos in vivo demonstram que as cepas de EPEC provocam profundas alteraçoes nas micro-vilosidades do enterócito, causando lesoes em pedestal. Objetivos. Neste trabalho, os autores relatam as alteraçoes da ultra-estrutura do intestino delgado causada pelos sorotipos 0111ab:H2, 0119:H6 e 018ab:H14, em lactentes portadores de diarréia aguda, e a capacidade destas bactérias de penetrar e se multiplicar no interior de células HeLa. Pacientes e Métodos. Estes sorotipos de EPEC foram isolados das fezes de três pacientes portadores de diarréia aguda que apresentaram intolerância alimentar e agravo nutricional. Alteraçoes ultra-estruturais do intestino delagado foram observadas nos espécimes estudados com formaçao de pedestal e penetraçao das bactérias no interior do citoplasma dos enterócitos. Estas bactérias também penetraram e se multiplicaram no interior das células HeLa. Conclusao. As alteraçoes ultra-estruturaisdescritas em pacientes com diarréria aguda devem ser levadas em conta quanto ao seu potencial efeito de provocar prolongamento da diarréia, seja por um fenômeno secretor ou por má absorçao dos nutrientes.

Vibrio fluvialis attachs to but does not enter Hela cell monolayers

Considering the possibility that invasiveness could be a neglected factor of virulence in Vibrio fluvialis-linked enteritis, since a dysenteric form of the disease was seen in Bangladesh, we studied 12 Brazilian strains of the organism, six clinical and six environmental, to determine whether they might be able to enter into HeLa cell monolayers or would carry plasmids incidentally involved in invasiveness. Four human and two environmental isolates attached to but did not enter into the cells. Though five strains harbored plasmids,no relationship was found between the carriage of these genetic elements and adhesiveness

Capacidade adesiva de amostras toxigênicas de Pasteurella multocida do tipo D em células HeLa, HEp-2 e de conchas nasais de suínos / Adhesive capability of toxigenic type D Pasteurella multocida strains in HeLa, HEp-2 and pig nasal turbinate cells

Três amostras toxigênicas de Pasteurella multocida foram submetidas a testes de aderência "in vitro", utilizando-se linhagens de células HeLa, HEp-2 e preparados de células de conchas nasais de suínos (CCNS). Para o teste de adesäo em CCNS, as amostras foram cultivadas em caldo infuso de cérebro e coraçäo, Agar Sangue (AS), meio de Minca com soro bovino e no meio de Heddlestone, com e sem alfa, alfa, dypiridil. Amostras de P. multocida (tipo D) näo aderiram em cultivos de células HeLa e HEp-2. Nos testes de aderência de P. multocida com CCNS, verificou-se que 80 por cento das células ciliadas continham menos do que três bactérias aderidas aos cílios ou ao corpo celular. Entre as células näo ciliadas, este número foi de 5 por cento. As amostras de Pasteurella multocida näo diferiram entre si e nem com relaçäo à E. coli-K12 (controle negativo) na capacidade adesiva. Pelos testes realizados "in vitro", concluiu-se que Pasteurella multocida possui pouca afinidade para as células testadas nas condiçöes discriminadas

Virulence of Yersinia spp. strains isolated from non-human sources

1. Strains of Yearsinia enterocolitica, Y. intermedia, Y frederiksenii and Y. kristensenii from non-human sources were examined for virulence factors. Four of these strains were positive for autoagglutination, three were calcium dependent at 37-C, three produced lipase, five had the ability to bind Congo red, six had pyrazinamidase activity, and two had a 42 MDa plasmid. 2. One strain (Y. enterocolitica 0:5) was lethal for mice and had the ability to invade guinea pig eyes and HeLa cells. After inoculation of mice by the intravenous route, this strain was isolated from the cecum. The spleen, liver, kidneys and lymph nodes presented necrosis. After intragastric inoculation, the strain was isolated from all of the organs and tissues examined. 3. Three of the remaining strains invaded HeLa cells but none caused guinea pig conjunctivitis. 4. After intragastric inoculation, all the strains were isolated from the cecum but disappeared between dys 3 and 6. After intravenous infection, three strains produced necrosis of the spleen and were more invasive, eliciting infection in various organs. The remaining strains caused hypertrophy and hyperplasua of Peyer's patches and/or lymph nodes. 5. These results indicate that Y. enterocolitica 0:5 can be considered as virulent as typical European Y. enterocolitica strains. The remaining strains perphaps induce a weak immune response independent of virulence factors