Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis

Abstract During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-β after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-β and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

Resumo Durante a inserção de implantes dentários partículas de titânio podem ser liberadas na região peri-implantar levando ao processo de osteólise e a associação com a bactéria pode exacerbar ainda mais a reação inflamatória. Entretanto, a associação de uma bactéria altamente invasiva da cavidade oral, Porphyromonas gingivalis (Pg) e partículas de titânio ainda não foi investigada. Este estudo avaliou a reação pró-inflamatória de macrófagos humanos em contato com micro e nanopartículas de titânio associada a lipopolissacarídeo P. gingivalis (PgLPS). As células THP-1 foram utilizadas e tratadas durante 12, 24 e 48 h nos 6 seguintes grupos: Controle (C), PgLPS (L); micropartículas (M); nanopartículas (N); PgLPS e micropartículas (LM); PgLPS e nanopartículas (LN). Em seguida foram realizados os seguintes ensaios: i) a viabilidade celular utilizando MTS, ii) a morfologia celular por MEV e iii) expressão do fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β) e interleucina 6 (IL-6) por qRT-PCR e ELISA. Como estatística foi realizado o teste ANOVA two-way seguido pelo teste de Tukey (p<0,05). Após o tratamento, as células apresentaram viabilidade e morfologia semelhantes, demonstrando que os tratamentos não foram capazes de induzir a morte celular. A expressão de genes foi significativamente mais elevada para o TNF-α e IL1-β após 12h, e para a IL-6 após 24 horas em N e grupos de LN. A produção de citocinas em relação ao tempo representou uma curva ascendente para o TNF-α com o pico em 48 h, enquanto que para IL1-β e IL-6 se apresentou como uma linha reta com relação ao tempo, exceto pelo grupo N que foi significativo para IL-6 em 48 h . Conclui-se, a partir destes resultados, que as nanopartículas de titânio produziram o maior estímulo na resposta pró-inflamatória nos macrófagos, independente da sua associação com LPS de P. gingivalis.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Infección por Brucella canis en humanos: propuesta de un modelo teórico de infección a través de la ruta oral / Brucella canis infection in humans: Proposal of a theoretical model of infection through the oral route

La infección por Brucella canis en los humanos se ha reconocido recientemente como una zoonosis, pero frecuentemente es sub reportada debido a que los síntomas pueden confundirse con los de un resfriado común u otras infecciones causadas por otros patógenos. Los caninos son los hospederos primarios de Brucella canis ; el incremento en la tendencia de tener perros como mascotas podría también aumentar la posibilidad de transmisión de la infección a los humanos por el estrecho contacto entre la mascota infectada y su propietario. En Colombia, hay reportes de aislamientos de B. canis de caninos de criaderos y de un humano en contacto con perros infectados, al igual que reportes de caninos seropositivos a la infección. Sin embargo, no hay mucha información disponible sobre los mecanismos de interacción hospedero-patógeno que conduzcan al establecimiento de la infección por Brucella canis en perros y en humanos esta información es todavía menor. En esta revisión se propone un modelo para la infección humana con Brucella canis a través de la ruta oral utilizando la información disponible para otras especies de Brucella que infectan al humano, incluyendo B. abortu s y B. melitensis , que difieren de B. canis en la composición estructural de su lipopolisacárido. También se hipotetiza el mecanismo de infección celular que es usado por B. canis para invadir y establecer la infección en células no fagocíticas y fagocíticas.

Brucella canis infection in humans has recently been recognized as a zoonosis, but it is frequently under reported because the flu-like symptoms are often confused with the presence of other disease-causing pathogens. Dogs are the primary hosts for Brucella canis ; the increasing trend to adopt dogs as pets also enhances the likelihood of transmission of Brucella canis infection through contact between infected dogs and owners. In Colombia, there are reports of isolates of B. canis from kennel dogs and also from one human being along with seropositive results from dogs and humans. However, the mechanism of hostpathogen interactions leading to the infection of Brucella canis in dogs is still unknown and even less is known about human infections. This review proposes a model for human infection with Brucella canis through the oral route. We use the information available for other human-infecting Brucella species, including B. abortu s and B. melitensis, which differ from B. canis in the structural composition of the lipopolysaccharide molecule. The mechanism of cellular infection used by B. canis to invade and establish infection in nonphagocytic and phagocytic cells is also hypothesized.

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen / Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.

Principales características y diagnóstico de los grupos patógenos de Escherichia coli / Principal characteristics and diagnosis of the pathogenic groups of Escherichia coli

Escherichia coli colonizes the human intestinal tract within hours of birth and is considered a non-pathogenic member of the normal intestinal flora. However, there are six pathogenic groups that may produce diarrhea: enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC) and diffusely adherent (DAEC) groups. E. coli can be isolated and classified using traditional methods, by identifying its biochemical or serum characteristics. The pathogenic mechanisms may be studied in cell cultures and animal model assays, as well as more up to date molecular biology methods for study and diagnosis. The latter have proven that genes are involved in pathogenesis. The objective of the present work is to draw attention to the importance of E. coli as a pathogenic organism. This microorganism is an etiologic agent of sporadic cases of diarrhea, hemorrhagic colitis, dysentery, and hemolytic uremic syndromes and outbreaks. Diarrheic E. coli manifestations occur mainly among infants, and deep knowledge and understanding of this microorganism are crucial to better epidemiologic surveillance.

Resistencia de diferentes sorogrupos de seratia marcescens ao soro humano normal e sua patogenicidade para camundongos / Serratia marcescens: studies on normal human serum resistance, serogrouping and pathogenicity for mice

Isolados nosocomiais de Serratia marcescens pertencentes a diferentes sorogrupos (01, 03, 04, 05, 010, 013, 016, 019 e 06,14) foram analisados quanto a resistencia à açäo bactericida do soro humano (HS). De 60amostras estudadas, 55 (91,6 por cento) foram HS-resistentes para uma concentraçäo de 50 (por cento) de soro e somente amostras do sorogrupo 06,14 foram HS-sensíveis. Todos os isolados de sangue, olhos e ossos foram resistentes ao HS. Amostras com diferentes sensibilidades ao HS foram testadas para a patogenicidade e todas foram letais para camundongos com a mesma DL50 (3,5 x 10(elevado a sétima potencia)). Esses resultados sugerem que a resistência ao soro näo é o principal determinante na virulência de S. marcescens.