Exploring tumourigenic potential of the parasite Anisakis: a pilot study.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.

Genotypic and phenotypic diversity in Enterococcus faecalis: is agar invasion a pathogenicity score? / Diversidad genotípica y fenotípica en Enterococcus faecalis: ¿Es la invasión en agar un marcador de patogenicidad?

Objectives. The main objective of the present study is to analyze different genotypic and phenotypic traits related to virulence in Enterococcus faecalis, as well as evaluated the agar invasion phenotype in a collection of isolates with different clinical origins. Material and methods. Seventy-nine E. faecalis isolates, with invasive and non-invasive clinical origins, have been used in this work. Presence of cytolysin activator (cylA), gelatinase (gelE), surface protein (esp), aggregation substance (asa1), endocarditis antigen (efaA), and collagen-binding protein (ace) have been analyzed by PCR. Phenotypic characterization included gelatinase activity, haemolysin production, biofilm formation and agar invasion. Results. All the isolates tested harboured at least one of the virulence determinants. The 95.5% of isolates from haematologic samples were positive for agar invasion test, significantly higher than isolates from non-invasive diseases. A significant reduction in relative invasion area was observed in three selected agar-invasive strains after 15 serial passages. Conclusions. It has been observed a significant high prevalence of agar-invasion positive isolates among strains belonged to haematological samples. Agar invasiveness is reduced after adaptation of clinical isolates to laboratory conditions, showing that agar invasion phenotype can be modulate by culture conditions as other virulence factors observed in different bacterial species (AU)

Objetivos. El principal objetivo de este trabajo es la caracterización de determinantes de virulencia genotípicos y fenotípicos relacionados con patogenicidad en Enterococcus faecalis, evaluando además el fenotipo de invasión en agar en una colección de aislados clínicos de diversa procedencia. Material y métodos. Se han analizado 79 cepas de E. faecalis aisladas en infecciones invasivas y no invasivas. La detección de los principales determinantes asociados a la virulencia (cylA, gelE, esp, asa1, efaA y ace) se ha realizado mediante PCR. La caracterización fenotípica incluyó la detección de actividad gelatinasa, hemólisis, formación de biofilm y el test de invasión en agar. Resultados. Todos los aislados presentaron, al menos, un determinante de virulencia. El 95,5% de las cepas provenientes de hemocultivos resultaron positivas para el test de invasión en agar, significativamente superior a lo observado en cepas de origen clínico no invasivo. En tres cepas seleccionadas, positivas para el test de invasión en agar, se observó una reducción significativa del área relativa de invasión tras 15 pases seriados. Conclusiones. Se ha observado una alta prevalencia de cepas con alto grado de invasión en agar en los aislados hematológicos. Dicho grado de invasión disminuye significativamente al adaptar tres cepas al crecimiento en condiciones de laboratorio, sugiriendo una modulación en función de las condiciones de cultivo tal y como ocurre con otros determinantes asociados a virulencia en diferentes especies bacterianas (AU)

[Structural dynamics of Legionella pneumophila and Legionella bozemanii colony/biofilm]. / Dinámica estructural de colonias/biofilm de Legionella pneumophila y Legionella bozemanii.

OBJECTIVES: The genus Legionella includes very pleomorphic species responsible for disease outbreaks in humans. The appearance of such has great importance to develop artificial biofilms in aquatic ecosystems. The aim of this work was to study the dynamics of growth and evolution of the internal structure of colonies of representative species of the genus as static biofilm model. METHODS: Isolated colonies of Legionella pneumophila and Legionella bozemanii grown in specific media for three and fifteen days were processed for histological methods and embedded in paraffin and epoxy resin for analysis by light microscopy, electron microscopy and image analysis. RESULTS. In colonies of both species were observed and defined specific architectural patterns, based on stratification and evolve over time. The strata differ in the amount of extracellular matrix, the morphology and population density and the proportion of dead cells. The internal structure of three days colonies showed large differences between L. pneumophila (two layers) and L. bozemanii (four layers). However, in the fifteen days colonies of both species evolved towards a common unique pattern formed by three layers. In both species the growth was also found within the culture medium, although this phenomenon was more intense in L. bozemanii with unique, central and larger invasions. CONCLUSIONS: Our results demonstrate that Legionella colonies on solid culture media are a good model of static biofilm with a complex structural dynamics characterized by the presence of morphological and functional subpopulations. We bring here an histological approach model, allowing, in further research, detailed studies in evolutionary adaptations in multicellular communities to adverse media and to antimicrobials in Legionella species of clinical interest.

Morphological plasticity of Streptococcus oralis isolates for biofilm production, invasiveness, and architectural patterns.

OBJECTIVE: Streptococcus oralis is an early coloniser of the oral cavity that contributes to dental plaque formation. Many different genotypes can coexist in the same individual and cause opportunistic infections such as bacterial endocarditis. However, little is known about virulence factors involved in those processes. The aim was to analyze the evolving growth of S. oralis colony/biofilm to find out potentially pathogenic features. DESIGN: Thirty-three S. oralis isolates were analyzed for: (1) biofilm production, by spectrophotometric microtiter plate assay; (2) colonial internal architecture, by histological methods and light and electron microscopy; (3) agar invasion, by a new colony-biofilm assay. RESULTS: S. oralis colonies showed two different growth patterns: (1) fast growth rate without invasion or minimally invasive; (2) slow growth rate, but high invasion ability. 12.1% of strains were biofilm non-producers and 24.2% not invasive, compared to 51.5% biofilm high-producers and 39.4% very invasive. Both phenotypic characteristics tended to be mutually exclusive. However, a limited number of strains (15%) co-expressed these features at the highest level. CONCLUSIONS: Morphological plasticity of S. oralis highlighted in this study may have important ecological and clinical implications. Coexistence of strains with different growth patterns could produce a synergic effect in the formation and development of subgingival dental plaque. Moreover, invasiveness might regulate dissemination and colonisation mechanisms. Simultaneous co-expression of high-invasive and high-biofilm phenotypes gives a fitness advantage during colonisation and may confer higher pathogenic potential.

Dinámica estructural de colonias/biofilm de Legionella pneumophila y Legionella bozemanii / Structural dynamics of Legionella pneumophila and Legionella bozemanii colony/biofilm

Objetivos. El género Legionella engloba especies muy pleomórficas responsables de brotes infecciosos en humanos. En la aparición de los mismos tiene gran importancia el desarrollo de biofilms en ecosistemas acuáticos artificiales. El objetivo de este trabajo fue estudiar la dinámica de crecimiento y la evolución de la estructura interna de colonias de especies representativas del género como modelo de biofilm estático. Material y métodos. Colonias aisladas de Legionella pneumophila y Legionella bozemanii crecidas en medios específicos durante tres y quince días fueron procesadas por métodos histológicos de inclusión en parafina y resina epoxi para su análisis mediante microscopía óptica, microscopía electrónica y análisis de imagen. Resultados. En las colonias de ambas especies se observaron patrones arquitecturales definidos y específicos, basados en la estratificación y que evolucionan en el tiempo. Los estratos se diferencian por la cantidad de matriz extracelular, la morfología y densidad poblacional y la proporción de células muertas. La estructura interna de las colonias de tres días presentaba grandes diferencias entre L. pneumophila (dos estratos) y L. bozemanii (cuatro estratos). Sin embargo, en las colonias de quince días ambas especies evolucionaron hacia un patrón único común formado por tres estratos. En ambas especies se comprobó también el crecimiento en el interior del medio de cultivo, aunque este fenómeno fue mucho más intenso en L. bozemanii, con invasiones únicas, centrales y de gran tamaño. Conclusiones. Nuestros resultados demuestran que las colonias de Legionella sobre medio de cultivo sólido son un buen modelo de biofilm estático, con una dinámica estructural compleja caracterizada por la presencia de subpoblaciones morfológicas y funcionales. La aproximación histológica empleada en este modelo permitirá estudiar adaptaciones evolutivas de comunidades multicelulares a medios hostiles, así como la respuesta a los antimicrobianos de las especies de Legionella de interés clínico (AU)

Objectives. The genus Legionella includes very pleomorphic species responsible for disease outbreaks in humans. The appearance of such has great importance to develop artificial biofilms in aquatic ecosystems. The aim of this work was to study the dynamics of growth and evolution of the internal structure of colonies of representative species of the genus as static biofilm model. Methods. Isolated colonies of Legionella pneumophila and Legionella bozemanii grown in specific media for three and fifteen days were processed for histological methods and embedded in paraffin and epoxy resin for analysis by light microscopy, electron microscopy and image analysis. Results. In colonies of both species were observed and defined specific architectural patterns, based on stratification and evolve over time. The strata differ in the amount of extracellular matrix, the morphology and population density and the proportion of dead cells. The internal structure of three days colonies showed large differences between L. pneumophila (two layers) and L. bozemanii (four layers). However, in the fifteen days colonies of both species evolved towards a common unique pattern formed by three layers. In both species the growth was also found within the culture medium, although this phenomenon was more intense in L. bozemanii with unique, central and larger invasions. Conclusions. Our results demonstrate that Legionella colonies on solid culture media are a good model of static biofilm with a complex structural dynamics characterized by the presence of morphological and functional subpopulations. We bring here an histological approach model, allowing, in further research, detailed studies in evolutionary adaptations in multicellular communities to adverse media and to antimicrobials in Legionella species of clinical interest (AU)

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm.

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms. This new methodology is simple, fast, reproducible, objective, inexpensive and can be used to study a great number of specimens simultaneously, all of which make it suitable for incorporation to the routine of any microbiology laboratory. It could also be a useful tool for additional studies related to clinical aspects of bacterial isolates such as virulence and antimicrobial response.

Histological approach to Bacillus subtilis colony-biofilm: evolving internal architecture and sporulation dynamics.

Bacillus subtilis has been used as a classic model to study biofilm formation and sporulation process. Colonies of wild-type strains usually have a complex external morphology, but the details of their internal architecture are still undisclosed. Since bacterial biofilms fulfill the criteria to be considered tissues, the aim of this work was to analyse B. subtilis colony-biofilm internal architecture evolution and sporulation dynamics using histological techniques. Transversal sections of colony-biofilms incubated from 24 hours up to 20 days were stained using histochemical techniques to analyse the internal structure by light and electron microscopy. A morphometric study of the different structural biofilm components was performed by image analysis, and an application to quantify spores was developed. Internal biofilm architecture was characterised by a stratified pattern, which evolved from 3 strata at 24 hours, up to 5 strata at 20 days. At 48 hours, strata at the central area of the biofilm was folded, resulting in elevated structures (vein-like structures) that could reach up to 465 µm in height. Sporulation started at 48 hours, at the top of the vein-like structures, at the interface between the two uppermost strata. At 20 days spores formed a continuous central layer, representing 7.5% of the total biofilm. In summary, our results demonstrate that B. subtilis colony-biofilm has a complex and organized internal architecture, evolving over time, and taking place in different cell subpopulations with different functionalities. Furthermore, in situ spore quantification described in this work could be a good alternative to the classical chamber counting.

Invasion of solid culture media: a widespread phenotypic feature of clinical bacterial isolates.

OBJECTIVES: The in-depth growth in solid culture media is a common feature in filamentous fungi and yeasts. However, there are very few bacterial species in which this phenomenon has been documented. The aim of this work was to assess the agar invasiveness of a wide range of Gram-positive and Gram-negative bacterial species of clinical interest. MATERIAL AND METHODS: Three different clinical isolates for each of eleven bacterial species were plated onto Columbia blood agar and let grow up to 15 days. Isolated colonies were processed by histological methods, embedded in epoxy resin, and then, semithin sections were stained with toluidine blue and visualized by light microscopy. RESULTS: Growth within the agar was observed in at least one strain in 9 of the 11 studied species. Invasions of Gram-negative rods were small, not plentiful, and round or triangle-shaped. In Gram-positive cocci, invasions were of big size, abundant and of variable shape (lentiform, globular, irregular, arrowhead) depending on the species. CONCLUSIONS: We propose that the growth within the agar can indicate a survival strategy common to many bacterial species, and so far, not previously reported. This strategy could be either a nutrient gradient tropism or the spread and colonization of new ecological niches, with potential implications in pathogeny.

Invasion of solid culture media: a widespread phenotypic feature of clinical bacterial isolates

Objetivos. El crecimiento en profundidad en medios de cultivo sólidos es un fenómeno habitual en hongos filamentosos y levaduras. Sin embargo, son muy escasas las especies bacterianas en las que se ha documentado este fenómeno. El objetivo de este trabajo fue comprobar la capacidad de invasión del agar de un amplio abanico de especies bacterianas grampositivas y gramnegativas de interés clínico. Material y métodos. Se cultivaron tres cepas de cada una de once especies bacterianas sobre agar Columbia hasta un máximo de 15 días. Colonias aisladas fueron procesadas e incluidas en resina epoxi por métodos histológicos y secciones transversales semifinas teñidas con azul de toluidina fueron visualizadas por microscopía óptica. Resultados. Se observó crecimiento en el interior del agar en al menos una de las cepas de nueve de las especies estudiadas. En bacilos gramnegativos, las invasiones eran escasas en número, pequeñas y con morfología redondeada o triangular. En cocos grampositivos las invasiones eran de gran tamaño, numerosas y de morfología variable (lentiforme, globular, irregular, en forma de punta de flecha, etc.) según la especie. Conclusiones. En nuestra opinión, el crecimiento en el interior del agar puede significar una estrategia de supervivencia común a muchas especies bacterianas y hasta ahora prácticamente no descrita. Esta estrategia podría ser el reflejo de un tropismo a favor de gradiente de nutrientes, o bien un fenómeno de diseminación y colonización de nuevos nichos ecológicos, con posibles implicaciones en la patogenia(AU)

Objectives. The in-depth growth in solid culture media is a common feature in filamentous fungi and yeasts. However, there are very few bacterial species in which this phenomenon has been documented. The aim of this work was to assess the agar invasiveness of a wide range of Gram-positive and Gram-negative bacterial species of clinical interest. Material and methods. Three different clinical isolates for each of eleven bacterial species were plated onto Columbia blood agar and let grow up to 15 days. Isolated colonies were processed by histological methods, embedded in epoxy resin, and then, semithin sections were stained with toluidine blue and visualized by light microscopy. Results. Growth within the agar was observed in at least one strain in 9 of the 11 studied species. Invasions of Gramnegative rods were small, not plentiful, and round or triangleshaped. In Gram-positive cocci, invasions were of big size, abundant and of variable shape (lentiform, globular, irregular, arrowhead) depending on the species. Conclusions: We propose that the growth within the agar can indicate a survival strategy common to many bacterial species, and so far, not previously reported. This strategy could be either a nutrient gradient tropism or the spread and colonization of new ecological niches, with potential implications in pathogeny(AU)

A rat model of intragastric infection with Anisakis spp. live larvae: histopathological study.

Anisakiasis is a fish-borne parasitic disease caused by consumption of raw or undercooked fish or cephalopods parasited by Anisakis spp. third stage larvae. The pathological effects of the infection are the combined result of the mechanical action of the larva during tissue invasion, the direct tissue effects of the excretory/secretory products released by the parasite, and the complex interaction between the host immune system and the Anisakis antigens. The aim of this study was to develop an experimental model of infection with Anisakis spp. live larvae in rats, useful to study the acute and chronic histopathological effects of the Anisakis infection. Sprague-Dawley rats were subjected to esophageal catheterization to place larvae directly into the stomach. Reinfections at different intervals after the first infection were preformed. Live larvae were found anchored to the mucosa and passing through the wall of the stomach and showed a strong resistance being able to stay alive at different sites and at the different pH. Migration of larvae from the stomach to other organs out of the gastrointestinal tract was also observed. The histopathological study showed the acute inflammatory reaction, with predominance of polymorphonuclear eosinophils and a mild fibrotic reaction. The model of infection described is valid to study the behavior of the larvae inside the host body, the histopathological changes at the invasion site, and the effects of the repeated infections by ingestion of live larvae.

Serotonin skews human macrophage polarization through HTR2B and HTR7.

Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT(1-7)) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization-associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT(7) mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT(2B) and 5HT(7) receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT(2B) was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT(2B) and 5HT(7), whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.

Effect of protein binding on the activity of voriconazole alone or combined with anidulafungin against Aspergillus spp. using a time-kill methodology.

OBJECTIVES: the aims of the study were to explore the activity of total and free (according to protein binding) maximal concentrations achieved in serum after multiple doses of voriconazole 400/200 mg and anidulafungin 200/100 mg against Aspergillus fumigatus and Aspergillus flavus and the human albumin or serum effects on antifungal activity. MATERIAL AND METHODS: Time-kill curves were performed with two A. fumigatus and two A. flavus strains at voriconazole and anidulafungin Cmax concentrations using different media: a) RPMI broth (Cmax-RPMI); b) RPMI with human serum (Cmax-HS), and c) RPMI with human albumin (Cmax-HAlb). In parallel, free-drug (fCmax) concentrations considering theoretical protein binding were performed in RPMI broth. Aspergillus metabolic activity was measured by the XTT reduction assay. RESULTS: Voriconazol or voriconazole plus anidulafungin reduced >88.4% the metabolic activity of Aspergillus sp. at Cmax-RPMI and fCmax after 48 h of exposition. Anidulafungin alone showed poor metabolic reductions (<80.1% at Cmax- RPMI and <15% at fCmax). Anidulafungin activity, but not voriconazole activity alone or combined decreased in presence of HS or HAlb (more pronounced in A. flavus strains and HAlb). However, anidulafungin Cmax-HS or Cmax-HAlb against A. fumigatus strains were significantly more active (p<0.05) than fCmax in RPMI. These species and culture medium-dependent impact of human protein binding in the activity of anidulafungin was related to macroscopic and microscopic differences among mycelial mat grown in RPMI, HS or HAlb in whose XTT retention was different. CONCLUSIONS: Synergism could not be demonstrated due to the high activity showed by voriconazole. Protein binding has not impact on voriconazole activity and this impact is considerably less than predicted by free concentration extrapolated from theoretical binding rate on anidulafungin. The XTT colorimetric assay needs to be standardized for use with Aspergillus spp. since without DMSO extraction the activity of echinocandins in a free-human protein RPMI medium could be overestimated.

Effect of protein binding on the activity of voriconazole alone or combined with anidulafungin against Aspergillus spp. using a time-kill methodology

Objetivos: los objetivos del estudio fueron valorar la actividad de las concentraciones máximas totales y libres (de acuerdo a la unión a proteínas) alcanzadas en suero después de dosis múltiples de voriconazol 400/200 mg y anidulafungina 200/100 mg frente Aspergillus fumigatus y Aspergillus flavus y el efecto del suero y albúmina humana en la actividad antifúngica. Material y métodos: las curvas de letalidad fueron realizadas con 2 cepas de A. fumigatus y 2 cepas de A. flavus a las concentraciones Cmax de voriconazol y anidulafungina empleando diferentes medios: a) caldo RPMI (Cmax-RPMI); b) RPMI con suero humano (Cmax-SH) y c) RPMI con albúmina humana (Cmax-AlbH). Se compararon con las concentraciones libres (fCmax) en caldo RPMI teniendo en cuenta la unión a proteínas. La actividad metabólica de las cepas de Aspergillus fue medida por la técnica de reducción de XTT. Resultados: voriconazol o voriconazol con anidulafungina redujeron la actividad metabólica de las cepas de Aspergillus >88.4% a las concentraciones Cmax-RPMI y fCmax después de 48 h de exposición. Anidulafungina sola mostró bajas reducciones metabólicas (<80.1% a Cmax-RPMI y <15% a fCmax). La actividad de anidulafungina, pero no la de voriconazol solo o combinado, disminuyó en presencia de suero o albúmina (más pronunciado para las cepas de A. flavus y con albúmina). Sin embargo, las concentraciones de anidulafungina Cmax-HS o Cmax-AlbH frente las cepas de A. fumigatus fueron significativamente más activas (p<0.05) que la concentración fCmax en RPMI. Este impacto de la unión a proteínas en la actividad de anidulafungina dependiente de la especie y el medio de cultivo fue relacionada con diferencias macroscópicas y microscópicas del crecimiento miceliar en RPMI, SH o AlbH en los cuales la retención fue diferente. Conclusiones: el sinergismo entre ambos antifúngicos no pudo ser demostrado probablemente por alta actividad mostrada por voriconazol. La unión a proteínas plasmáticas no tiene impacto en la actividad de voriconazol y su efecto es considerablemente menor sobre anidulafungina que el pronosticado por la concentración libre teórica extrapolada de la tasa de unión a proteínas. El método colorimétrico del XTT necesita ser estandarizado para ser empleado con Aspergillus sp. ya que sin la extracción con DMSO la actividad de las equinocandinas en el medio RPMI sin proteínas humanas podría ser sobreestimada(AU)

Objectives: the aims of the study were to explore the activity of total and free (according to protein binding) maximal concentrations achieved in serum after multiple doses of voriconazole 400/200 mg and anidulafungin 200/100 mg against Aspergillus fumigatus and Aspergillus flavus and the human albumin or serum effects on antifungal activity. Material and methods: Time-kill curves were performed with two A. fumigatus and two A. flavus strains at voriconazole and anidulafungin Cmax concentrations using different media: a) RPMI broth (Cmax-RPMI); b) RPMI with human serum (Cmax-HS), and c) RPMI with human albumin (Cmax- HAlb). In parallel, free-drug (fCmax) concentrations considering theoretical protein binding were performed in RPMI broth. Aspergillus metabolic activity was measured by the XTT reduction assay. Results: Voriconazol or voriconazole plus anidulafungin reduced >88.4% the metabolic activity of Aspergillus sp. at Cmax-RPMI and fCmax after 48 h of exposition. Anidulafungin alone showed poor metabolic reductions (<80.1% at Cmax- RPMI and <15% at fCmax). Anidulafungin activity, but not voriconazole activity alone or combined decreased in presence of HS or HAlb (more pronounced in A. flavus strains and HAlb). However, anidulafungin Cmax-HS or Cmax-HAlb against A. fumigatus strains were significantly more active (p<0.05) than fCmax in RPMI. These species and culture medium-dependent impact of human protein binding in the activity of anidulafungin was related to macroscopic and microscopic differences among mycelial mat grown in RPMI, HS or HAlb in whose XTT retention was different. Conclusions: Synergism could not be demonstrated due to the high activity showed by voriconazole. Protein binding has not impact on voriconazole activity and this impact is considerably less than predicted by free concentration extrapolated from theoretical binding rate on anidulafungin. The XTT colorimetric assay needs to be standardized for use with Aspergillus spp. since without DMSO extraction the activity of echinocandins in a free-human protein RPMI medium could be overestimated(AU)

Liver impairment after portacaval shunt in the rat: the loss of protective role of mast cells?

Mast cells are involved in various liver diseases and appear to play a broader pathogenic role than originally thought. They may participate in the splanchnic alterations related to a porto-systemic shunt. To verify this hypothesis we studied the serum and hepatic histological changes in rats four weeks after an end-to-side portacaval shunt. In this experimental model of chronic liver insufficiency we also assessed the mucosal mast cells (MMC) and connective tissue mast cells (CTMC) in the liver, mesenteric lymph nodes and small intestine, as well as the serum levels of rat mast cell protease-II (RMCP-II). The results show liver and testes atrophy, with hypoalbuminemia (p=0.0001), hyperbilirubinemia (p=0.0001) and increase in aspartate aminotransferase (p=0.004) and alanine aminotransferase (p=0.0001). Hepatic histopathology demonstrates hepatocytic necrosis and apoptosis, portal inflammation, biliary proliferation, steatosis and fibrosis. There is a decrease of MMCs and CTMCs in the liver, while in the ileum CTMCs increase and MMCs decrease. These results suggest the involvement of mast cells in the pathophysiological splanchnic impairments in this experimental model. In particular, the decreased number of liver mast cells may be associated with the hepatic atrophy. If this is the case, we propose that the disruption of the hepato-intestinal axis after a portocaval shunt in the rat could inhibit the ability of the liver to developing an appropriate repair response mediated by mast cells.

Hypodermal decidualized endometrioma with aberrant cytokeratin expression. A lesion mimicking malignancy.

Decidualized endometrioma is a pseudoneoplastic lesion that may appear as a solitary nodule in the hypodermis, simulate a malignant epithelioid tumor, and can represent a diagnostic challenge. A 36-year-old woman delivered a full-term baby by cesarean. At the immediate puerperium, she complained of a subcutaneous nodule measuring 2.5 cm, underneath a previous caesarean scar from the former full-term delivery 3 years earlier. Histologic features included a nodular growth pattern of large monomorphic epithelioid cells showing diffuse positivity for cytokeratin (AE1/AE3, 18), human placental lactogen, and CD10 and focal positivity for inhibin alpha. The main differential diagnoses include trophoblastic neoplasia and deciduoid mesothelioma. Good clinicopathological correlation is essential for the correct diagnosis. Immunohistochemical stains can be misleading. An important clue is the combination of large decidualized cells and lumens lined by flat or low cuboidal cells that are atrophic endometrial glands. This lesion has a benign behavior.

Dendritic cell-specific ICAM-3-grabbing nonintegrin expression on M2-polarized and tumor-associated macrophages is macrophage-CSF dependent and enhanced by tumor-derived IL-6 and IL-10.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF-inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14(+) CD163(+) IL-10-producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewis(x)-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4-dependent) and regulatory (M-CSF-dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.

Is infection by Dermatophilus congolensis underdiagnosed?

Dermatophilus congolensis, which affects animal species, is an uncommon human infection. Few cases, mainly in tropical areas, have been reported. We describe the first human infection in Spain in a traveler returning from Central America. Diagnosis of human infection may be underestimated in people in contact with animals.

Splanchnic Th(2) and Th(1) cytokine redistribution in microsurgical cholestatic rats.

BACKGROUND: Long-term extrahepatic cholestasis in the rat induces ductular proliferation and fibrosis in the liver, portal hypertension, splenomegaly, portosystemic collateral circulation, and ascites. These splanchnic alterations could have an inflammatory pathophysiology. MATERIAL AND METHODS: We measured serum levels of hepatobiliary injury markers and the acute phase proteins, alpha-1-major acid protein (alpha(1)-MAP) and alpha-1-acid glycoprotein (alpha(1)-GPA) in rats 6 wk after microsurgical extrahepatic cholestasis. We also assayed Th(1) (TNF-alpha and IL-1beta) and Th(2) (IL-4 and IL-10) cytokine levels in the liver, ileum, spleen, and mesenteric lymph complex by enzyme-linked immunosorbent assay (ELISA) techniques. Liver fibrosis was measured by Sirius red stain and by using an image system computer-assisted method and mast cell liver infiltration by Giemsa stain. RESULTS: The cholestatic rats showed an increase (P<0.001) in serum levels of bile acids, total and direct bilirubin, AST, ALT, AST/ALT index, gamma-GT, alkaline phosphatase, alpha(1)- MAP, alpha(1)-GPA, and LDH (P<0.05) in relation to sham-operated rats. TNF-alpha, IL-1beta, IL-4, and IL-10 increased in the ileum (P<0.01) and mesenteric lymph complex (P<0.001), and decreased in the liver (P<0.001). A marked bile proliferation associated with fibrosis (P<0.001) and mast cell infiltration was also shown in the liver of cholestatic rats. CONCLUSION: The splanchnic redistribution of cytokines, with an increase of Th(1) and Th(2) production in the small bowel and in the mesenteric lymph complex, supports the key role of inflammatory mechanisms in rats with secondary biliary fibrosis.

Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages.

Macrophage activation comprises a continuum of functional states critically determined by cytokine microenvironment. Activated macrophages have been functionally grouped according to their response to pro-Th1/proinflammatory stimuli [lipopolysaccharide, IFNgamma, granulocyte macrophage colony-stimulating factor (GM-CSF); M1] or pro-Th2/anti-inflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2]. We report that folate receptor beta (FRbeta), encoded by the FOLR2 gene, is a marker for macrophages generated in the presence of M-CSF (M2), but not GM-CSF (M1), and whose expression correlates with increased folate uptake ability. The acquisition of folate uptake ability by macrophages is promoted by M-CSF, maintained by IL-4, prevented by GM-CSF, and reduced by IFNgamma, indicating a link between FRbeta expression and M2 polarization. In agreement with in vitro data, FRbeta expression is detected in tumor-associated macrophages (TAM), which exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRbeta is expressed, and mediates folate uptake, by CD163(+) CD68(+) CD14(+) IL-10-producing TAM, and its expression is induced by tumor-derived ascitic fluid and conditioned medium from fibroblasts and tumor cell lines in an M-CSF-dependent manner. These results establish FRbeta as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of therapeutic drugs are a potential immunotherapy tool to target TAM.

Colonial architecture and growth dynamics of Staphylococcus aureus resistant to methicillin

El objetivo del estudio fue observar la estructura colonialy la dinámica del crecimiento de Staphylococcus aureusresistente a meticilina (SARM) empleando cortes semifinosvisualizados al microscopio óptico. Se estudiaron unacepa de S. aureus sensible a meticilina (SASM) y un SARM.Después de cada periodo de incubación (24 y 48 h) a 37ºClas colonias fueron incluidas en una resina epoxi. Cortessemifinos de 0.5 μm fueron teñidos con azul de toluidina yvisualizados al microscopio. Desde el punto de vista microscópico,no se observaron diferencias estructurales entrelas colonias de SASM y SARM. Si se observaron diferenciasen ambas cepas entre las colonias de 24 y 48 h de incubación.En las colonias de 24 h se observaron 2 capasclaramente diferenciadas: (A) una capa basal con alta densidadde población en contacto con el medio de cultivo y(B) una capa superficial con menor densidad de población.En las colonias de 48 h se observaron cuatro capas: (A)una capa basal con alta densidad de población; (B) una capaclara constituida por restos bacterianos muy degradadosen cuyo seno se observan muy escasos y dispersos cocosque conservan sus propiedades tintoriales; (C) una capamixta, constituida por una mezcla de bacterias vivas y restosbacterianos muy groseros y poco degradados y (D) unacapa superficial con menor densidad de población que lacapa basal. Las colonias forman estructuras altamente organizadasoriginadas por la disponibilidad de nutrientes ymecanismos de comunicación intercelular. La arquitecturacolonial es un proceso complejo y tiempo dependiente(AU)

The aim of the study was to explore the structure andgrowth dynamics of Staphylococcus aureus resistant to methicillin(MRSA) colonies using semithin sections visualizedby light microscope. One S. aureus susceptible to methicillin(MSSA) and one MRSA clinical strains were studied. Coloniesin agar plates were embedded in epoxy resin after each incubationperiod (24 h and 48 h) at 37ºC. Semithin sections of0.5 μm were stained with toluidine blue and visualized by lightmicroscope. Microscopically, no structural differences wereobserved between SASM and SARM colonies but differenceswere observed in both strains between 24 and 48 h incubationperiods. Colonies showed two layers clearly differentiated at24 h independently of the resistance to methicillin: (A) onebasal layer with high density of population in contact withculture media, and (B) one superficial layer with a lower densityof population. Colonies showed four layers at 48 h: (A) onebasal layer with high density of population; (B) one clear layerconstituted by very degraded bacterial remains in which canbe observed cocci dispersed with their dyeing properties; (C)one mixed layer constituted by viable bacteria and little degradedbacterial remains (D) one superficial layer with a lowerdensity of population than basal layer. Colonial architecture isa complex and time-dependent process(AU)