Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus.

The present study evaluated the antimicrobial activity of three root canal irrigants against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. These microorganisms were incubated in the presence of citric acid (6 and 10%), EDTA (17%), and NaOCl (0.5, 1.0, 2.5, and 5.25%). Agar diffusion tests were performed and redox indicator resazurin was used to evaluate the inhibitory effect of the irrigants on the metabolic activity of these microorganisms. The mean diameters of the inhibition zones for the C. albicans cultures were 11.6 mm (17% EDTA), 5.5 mm (0.5% NaOCl), 12.9 mm (1% NaOCl), 22.1 mm (2.5% NaOCl), and 28.5 mm (5.25% NaOCl). The mean diameters of the inhibition zones for E. faecalis were 2.8 mm (1% NaOCl), 5.4 mm (2.5% NaOCl), and 8.3 mm (5.25% NaOCl). For S. aureus, the mean values were 8.0 mm (17% EDTA), 3.0 mm (1% NaOCl), 8.8 mm (2.5% NaOCl), and 10.0 mm (5.25% NaOCl). Most of the irrigant solutions presented effective antimicrobial activity against C. albicans. A high inhibitory effect on the metabolic activity of E. faecalis was detected when the microorganisms were incubated with 17% EDTA. The same result was reached when S. aureus was incubated in the presence of > 2.5% NaOCl. Altogether, these results indicate that 2.5% and 5.25% NaOCl are microbicides against S. aureus while 0.5% and 1% NaOCl are only microbiostatic against the tested bacteria. The 6% and 10% citric acid as well as 17% EDTA did not affect the viability of any of the assayed microorganisms.

Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus

The present study evaluated the antimicrobial activity of three root canal irrigants against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. These microorganisms were incubated in the presence of citric acid (6 and 10 percent), EDTA (17 percent), and NaOCl (0.5, 1.0, 2.5, and 5.25 percent). Agar diffusion tests were performed and redox indicator resazurin was used to evaluate the inhibitory effect of the irrigants on the metabolic activity of these microorganisms. The mean diameters of the inhibition zones for the C. albicans cultures were 11.6 mm (17 percent EDTA), 5.5 mm (0.5 percent NaOCl), 12.9 mm (1 percent NaOCl), 22.1 mm (2.5 percent NaOCl), and 28.5 mm (5.25 percent NaOCl). The mean diameters of the inhibition zones for E. faecalis were 2.8 mm (1 percent NaOCl), 5.4 mm (2.5 percent NaOCl), and 8.3 mm (5.25 percent NaOCl). For S. aureus, the mean values were 8.0 mm (17 percent EDTA), 3.0 mm (1 percent NaOCl), 8.8 mm (2.5 percent NaOCl), and 10.0 mm (5.25 percent NaOCl). Most of the irrigant solutions presented effective antimicrobial activity against C. albicans. A high inhibitory effect on the metabolic activity of E. faecalis was detected when the microorganisms were incubated with 17 percent EDTA. The same result was reached when S. aureus was incubated in the presence of > 2.5 percent NaOCl. Altogether, these results indicate that 2.5 percent and 5.25 percent NaOCl are microbicides against S. aureus while 0.5 percent and 1 percent NaOCl are only microbiostatic against the tested bacteria. The 6 percent and 10 percent citric acid as well as 17 percent EDTA did not affect the viability of any of the assayed microorganisms.

Citotoxidade de diferentes concentrações de hipoclorito de sódio sobre osteoblastos humanos / Citotoxicity of different amounts of sodium hypochlorite on human cultured osteoblasts

Objetivo: Avaliar o efeito citotóxico de diferentes concentrações de hipoclorito de sódio sobre uma cultura de células de osteoblastos humanos (linhagem HOB). Métodos: Culturas confluentes de osteoblastos humanos (linhagem HOB) foram obtidas em meio de Dulbecco modificado, suplementado com 10% de soro fetal bovino e submetidas a incubações com hipoclorito de sódio (concentrações de 0,5; 1,0; 2,5 e 5,25%) durante trinta segundos. O grupo controle foi representado por células incubadas em fosfato de potássio. A avaliação da viabilidade celular foi realizada através do teste de exclusão com azul de Trypan, em triplicata. Durante o período de incubação, imagens foram registradas através de um microscópio óptico invertido, para avaliação da morfologia celular. Resultados: Verificou-se que no grupo controle havia 98,7% de células viáveis, morfologicamente normais, enquanto que nos grupos experimentais, células viáveis não foram observadas. A cinética de citotoxidade seguiu tendência dependente da concentração. Conclusão: O hipoclorito de sódio nas concentrações 0,5; 1,0; 2,5; 5,25%, incubado por trinta segundos em cultura de osteoblastos humanos é citotóxico.

Objective: To evaluate the cytotoxic effect of different amounts of sodium hypochlorite, on a culture of human osteoblastos (HOB) cells. Method: Cultures of human osteoblasts (HOB) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of bovine fetal serum were incubated in sodium hypochlorite (concentrations of 0.5; 1.0; 2.5 and 5.25%) for thirty seconds. The control group was represented by cells incubated in phosphate buffered saline (PBS). Cell viability was assessed by means of 0.4% trypan blue dye exclusion test, in triplicate. During the incubation period, images were recorded through an inverted optic microscope to evaluate the cellular morphology. Results: In the control group 98.7% of viable cells were verified, without morphology alterations, while no viable cells were observed in the experimental groups. The kinetics of cytotoxity was concentration-dependent. Conclusion: It was concluded that there was a cytotoxic effect on cultures of human osteoblasts incubated for thirty seconds in sodium hypochlorite in all concentrations (0.5; 1.0; 2.5 and 5.25%).

A interface célula-matriz extracelular-biomaterial e a biocompatibilidade de implantes de titânio / The cell-extracellular matrix-biomaterial interface and the biocompatibility of titanium implants

O conhecimento acumulado na última década sobre as interações mecanoquímicas entre implantes de titânio e tecidos animais revela o papel crucial desempenhado pela interface célula-matriz extracelular (MEC)-titânio na biocompatibilidade do último. Nesse contexto, e focalizando a interação células ósseas-MEC-titânio, esta breve revisão discute e apresenta alguns dados originais relativos ao comportamento de células formadoras de osso ou osteoblastos sobre superfícies de titânio, sob a intermediação de componentes da matriz extracelular. Como enfatizado em publicações recentes, o conhecimento subjacente a este tema é relevante para as modernas clínicas odontológica, médica e veterinária, assim como para empresas envolvidas no desenho e no desenvolvimento de implantes de titânio.

The accumulated knowledge during last decade on the mechanochemical interactions between titanium implants and animal tissues has point out the crucial role played by the cell-extracellular matrix (ECM)-titanium interface in the biocompatibility of the last. In this context, and specifi cally focusing the bone cells-ECM-titanium interaction relationships, this brief revision includes a discussion and some original data all concerning the behavior of bone-forming cells or osteoblasts onto titanium surfaces under the intervention of extracellular matrix components. As emphasized in recent publications, the knowledge underlying this subject is relevant for the modern dental, medical and veterinary clinics, as well as to companies involved in both design and development of titanium implants.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis.

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

The binding of Tritrichomonas foetus to immobilized laminin-1 and its role in the cytotoxicity exerted by the parasite.

The recognition and binding of pathogens to extracellular matrix glycoproteins may determine the outcome of infective processes. The interaction between the bovine urogenital parasite Tritrichomonas foetus and the major basal membrane glycoprotein laminin-1 (LMN-1) was investigated. The chemical nature of parasite molecules involved in the attachment of T. foetus to immobilized LMN-1 and the influence of LMN-1 in the toxicity exerted by the parasite to HeLa cells was studied. Attachment of T. foetus to LMN-1 resulted in notable morphological alterations of the parasite, which became amoeboid. T. foetus recognized LMN-1 through specific amino acid sequences (AG73, C16, A208 and A13) in the LMN-1 molecule, and the protein nature of the parasite molecules involved in the recognition was demonstrated by dot-blot analyses. Such molecular recognition was cation-dependent and five LMN-1-binding molecules (220, 200, 130, 125 and 80 kDa) were identified in T. foetus. Binding of T. foetus to LMN-1 rendered the parasite toxic to HeLa cell monolayers. Thus, LMN-1 appears to provide signalling cues that mediate important cell functions in T. foetus concerning its interaction with host cells.

A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis.

Iron is an essential element to support the growth and survival of Trichomonas vaginalis. It plays a critical role in the host-parasite interaction, and modulates the expression of virulence factors in this protozoan. In this work, parasites grown in iron-rich and iron-depleted media were analyzed by (i) light and scanning electron microscopy and (ii) 2-DE and MS. Withdrawal of iron from the culture medium resulted in dramatic changes in both the morphology and in the proteome pattern of T. vaginalis. Trophozoites underwent transformation from ellipsoid or amoeboid forms to rounded cells, whose flagella and axostyle were internalized. Forty-five proteins differentially expressed in parasites cultivated in the absence of iron were identified. In iron-depleted parasites, enzymes involved in energetic metabolism, proteolysis and hydrogenosomal iron-sulfur (Fe-S) proteins were down-regulated or even suppressed. Among up-regulated proteins, six isoforms of actin were detected. In addition, phosphoenolpyruvate carboxykinase, putative lactate dehydrogenase, and putative adenosine triphosphatase were also up-regulated or were exclusively observed in gels related to iron-depleted parasites. Our data demonstrate that iron has a pivotal role in the regulation of the morphological transformation of T. vaginalis and modulates the expression of both Fe-S and non-Fe-S proteins in the parasite.

The interaction between Acanthamoeba polyphaga and human osteoblastic cells in vitro.

Acanthamoeba spp. contains a group of free-living amoebae widespread in nature. These microorganisms may cause several diseases in humans including osteomyelitis. Here we characterize the cellular interaction between clinical and freshwater isolates of A. polyphaga with human osteoblasts. Amoeba cytoadherence was evaluated quantitatively and qualitatively. We observed that the clinical isolate readily adheres to human osteoblastic cells (HOB) in a saturable and time-dependent fashion. The cytoadhesion appears to be in part dependent on mannose-associated surface glycoconjugates, since prior incubation of the amoebae with alpha-mannose reduced cytoadhesion approximately 75%. Scanning electron microscopy revealed various amoebae exhibiting acanthapodia contacting the surface of osteoblasts. Some osteoblasts developed morphologies resembling apoptotic cells. The clinical isolate was highly toxic to HOB cells during 24 h of cell-protozoan interaction. Cytotoxicity was also dependent on the amoeba-cell ratio. During the cytopathogenic process we observed amoebae in the apparent process of ingestion of target cells and also amoebae extending projections or digipodia into osteoblast targets. The results indicate that A. polyphaga trophozoites attach and destroy human osteoblasts.

Strategies by which some pathogenic trichomonads integrate diverse signals in the decision-marking process

The interaction between each one of Trichomonas vaginalis and Tritrichomonas foetus with their hosts is a complex process in which components associated to the cell surfaces of both parasites and host epithelial cells, and also to soluble components found in vaginal/urethral secretions, are involved. Either cytoadhesion or the cytotoxicity exerted by parasites to host cells can be dictated by virulence factors such as adhesins, cysteine proteinases, laminin-binding proteins, integrins, integrin-like molecules, a cell detachment factor, a pore-forming protein, and glycosidases among others. How trichomonads manipulate informations from the extracellular medium, transduce such informations, and respond to them by stimulating the activities of some surface molecules and/or releasing enzymes are the aspects concerning trichomonal virulence which are here briefly reviewed and discussed.

A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction

Basic surface properties of mononuclear cells from Didelphis marsupialis

The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measuments of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2 per cent after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells posses cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5º and 40.8º, respectively. Treatment of the cells, with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsable for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

Estudo do comportamento clínico e a microscopia eletrônica de cepas de Trichomonas vaginalis na Cidade do Rio de Janeiro / Clinical and scanning electronic microscopic behavior of Trichomonas vaginalis strains isolated on Rio de Janeiro city

O objetivo deste trabalho foi realizar um estudo sobre a tricomoníase no Rio de Janeiro, analisando o comportamento clínico de 20 portadores e, à microscopia eletrônica, de cinco cepas recém-colhidas de Trichomonas vaginalis, comparando os resultados finais com os observados na literatura mundial. Vinte pacientes com tricomoníase foram examinadas, a fim de caracterizá-las de acordo com a sintomatologia apresentada. A partir de secreçöes vaginais de algumas destas pacientes, foram colhidas cinco amostras deste protozoário para posterior análise microscópica em grande aumento. Alguns aspectos ginecológicos foram mais freqüentes na nossa casuística do que n a literatura internacional: odor fétido, dispareunia, desconforto abdominal baixo, secreçåo bolhosa e menor teor inflamatório local. A microscopia eletrônica foi observada presença de uma capa envoltória espessa na superfície celular parasitária e aparelho de Golgi bastante desenvolvido, inexistentes em cepas de cultivo laboratorial prolongado. Podemos atribuir essas diferenças estatísticas a fatores próprios do parasita, ao seu meio ambiente circundante e à resposta imunológica do hospedeiro, alertando-nos para a existência, em nosso meio, de cepas de T. vaginalis de patogenicidade superior àquelas deswcritas por autores diversos em outras regiöes do mundo, potencialmente indutoras de lesöes pré-neoplásicas na populaçåo feminina