Untersuchungen zur hBD-2- und hBD-3-Expression und -Regulation während einer Candida-Ösophagitis

Die epithelialen Abwehrmechanismen regulieren die Besiedlung der Epithelien durch Kommensale und überwachen das Wachstum pathogener Mikroorganismen, wie z.B. des opportunistischen Hefepilzes Candida albicans im Ösophagus. In Folge dessen kommt es bei gesunden Individuen selten zu einer übermäßigen Besiedlung des Ösophagusepithels durch Candida albicans oder zu einer Infektion (Candida-Ösophagitits). Bei alten Menschen oder immunsupprimierten Patienten entwickelt sich jedoch häufig eine Candida-Ösophagitis, die sich in einer lebensbedrohlichen Candida-Sepsis manifestieren kann. Die humanen -Defensine sind wichtige Vermittler der epithelialen Abwehr und werden während einer Candida-Ösophagitis in den Epithelzellen signifikant induziert, wie in Vorversuchen gezeigt werden konnte. Um die Signaltransduktionskaskaden untersuchen zu können, die für die Defensininduktion nach Candida albicans-Infektion des Ösophagusepithels verantwortlich sind, wurde ein in vitro Modell mittels der oesophagealen Zelllinie OE21 etabliert. Die Analysen ergaben, dass die Inkubation mit lebenden C. albicans-Zellen oder C. albicans-Überständen zu einer mäßigen Induktion der -Defensine hBD-2 und hBD-3 führt. HBD-1 wird weiterhin konstant exprimiert und konnte auch in den nachfolgenden Experimenten nicht induziert werden. Die Inkubation mit Candida albicans führt zur Aktivierung der Transkriptionsfaktoren NFB und AP-1. Die Kostimulation der OE21-Zellen mit Candida albicans und polymorphonukleären Zellen (PMZ) wirkt synergistisch auf die hBD-2- und hBD-3-Expression und induziert diese signifikant. Dieser synergistische Effekt führt ebenfalls zu einer verstärkten nukleären Translokation der Transkriptionsfaktoren NFB und AP-1. Mit Inhibitionsversuchen konnten die entscheidenden Signalkaskaden identifiziert werden. Dabei zeigte sich, dass der hBD-2-Promotor in Abhängigkeit von NFB und MAPK/AP-1 reguliert wird. Zudem deuten die Ergebnisse darauf hin, dass proinflammatorische Zytokine für die Induktion der hBD-2-mRNA-Transkription von entscheidender Bedeutung sind. Die Expression von hBD-3 erfolgt in Abhängigkeit von dem EGF-Rezeptor und ebenfalls über die MAP Kinasen und den Transkriptionsfaktor AP-1. Diese unterschiedliche Regulation der -Defensine ermöglicht eine gestaffelte Reaktion der Epithelien auf Mikroorganismen und Infektionen. HBD-1 kontrolliert das Wachstum der Kommensalen im gesunden Organismus, während hBD-2 und hBD-3 bei Infektionen aktiviert werden. Dabei könnte hBD-2 während der initialen Entzündungsphase als Bestandteil der antimikrobiellen Abwehr wirken, während hBD-3 zeitlich versetzt aktiviert wird und bei Reparaturprozessen und der Wundheilungsphase den Schutz der Epithelien übernehmen könnte.Epithelial barriers protect individuals against pathogen microorganisms and control growth of commensals. Commensals, like Candida albicans, are generally present in the oro-gastrointestinal tract but they do not reach significant numbers nor are of any clinical relevance to a healthy individual. However, in the oesophagus of immune compromised individuals or geriatric people a Candida oesophagitis is of frequent occurrence. Human -defensins are critical components of host defence at mucosal surfaces. Our recently published results showed that hBD-2 and hBD-3 are up-regulated in Candida oesophagitis. Therefore the role of several candidate signalling pathways in Candida albicans induced -defensin expression in the oesophageal cell line OE21 were studied and the potential role of polymorphonuclear cells (PMZ) in Candida oesophagitis were analysed. Real time RT-PCR analysis showed that in human oesophageal cell cultures Candida albicans is a very weak direct inducer of human -defensin mRNA, but the induction of hBD-2- and hBD-3-mRNA expression is strongly enhanced when OE21 cells were additionally cultured with PMZ. Gel shift analysis revealed that Candida albicans activated NFB and AP-1 signalling pathways in the oesophageal cell line. EGF receptor inhibition directly reversed the AP-1 activation and hBD-3 induction, indicating that the EGF receptor and the transcription factor AP-1 are crucial for hBD-3 expression. In addition, an intact MAP kinase pathway is necessary for hBD-3 and also for hBD-2 induction. In contrast to hBD-3, hBD-2 activation seems to be more reliable on cytokine induced NFB dependent signalling after exposure to Candida albicans cells. In oesophageal cell cultures AP-1 dependent pathways are also of importance for hBD-2 induction. HBD-1 expression is not affected by one of the tested stimuli. Its main function is the control of microbiota in the healthy organism, whereas hBD-2 and hBD-3 are not expressed at significant levels. The divergent signalling events regulating hBD-2 and hBD-3 expression suggest temporal functional variation in terms of infections. HBD-2 may contribute to antimicrobial barrier function during inflammatory phase while hBD-3 plays a more important role during repair and wound healing phase of infection

Funding Era Free Speech Theory: Applying Traditional Speech Protection to the Regulation of Anonymous Cyberspace

Abstract Background Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability. Methods Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d. Results A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1β (Interleukin-1β), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture. Conclusions The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions

Ontogenetic conflicts and rank reversals in two Mediterranean oak species: Implications for coexistence

In heterogeneous environments, species segregate spatially in response to selective abiotic and biotic filters occurring throughout plant ontogeny. Ontogenetic conflicts in recruitment may lead to spatially discordant patterns of regeneration among microhabitats with different plant cover. In addition, species differing in seed size may be subjected to opposing ecological and evolutionary pressures throughout the life cycle of the plant. We used a multi-stage demographic approach aimed at characterizing the main stage-specific probabilities of recruitment (seed survival, seed germination, seedling emergence and survival during the first 3years of life) in two Mediterranean oak species coexisting at southern Spain. We calibrated linear and nonlinear likelihood models for each of these consecutive life history stages and calculated overall probabilities of recruitment along a wide range of plant cover and seed size variation. Seed predation and seedling mortality over the dry season were the most limiting processes for the two studied oak species. However, species ranking diverged substantially through the life history stages considered in this study due to different ontogenetic trends among species. At the intraspecific level, recruitment-driving processes during the seed and the seedling stages showed opposing tendencies along the explored range of plant cover and seed size. Thus, small-sized acorns and open areas were favoured for the seed stage, whereas large acorns and dense microhabitats did for the seedling stage. The existence of opposing selective pressures on seed mass and their differential influence on the two studied oak species determined the occurrence of species-specific optimal seed sizes (small acorns for Quercus canariensis vs. acorns of large or intermediate size for Quercus suber). The spatial patterns predicted by our overall-recruitment models provided some evidence of regeneration niche partitioning in the two coexisting oak species, supporting their current distribution patterns as saplings and adults at the study area. Synthesis. We conclude that within- and among-species differences through plant ontogeny, arising from species differential response to microhabitat heterogeneity and seed size variation, could be of great importance for oak species niche segregation, driving stand dynamics and spatial pattern distribution along the landscape. The information provided by this study could be also applied to optimize management and restoration programmes since it has enabled us to identify the most favourable conditions and traits for recruitment in oak species that exhibit serious constraints for natural regeneration.This study was supported by a JAE-doc—contract to IMPR, by the Spanish MEC projects Heteromed (REN2002-4041-C02-02), Dinamed (CGL2005-5830-C03-01) and Interbos (CGL2008-04503-C03-01), the Andalusian PE2010-RNM-5782 project, and by European FEDER funds. This research is part of the Globimed (http:// www.globimed.net) network in forest ecology.Peer Reviewe

Legionella pneumophila induces human beta Defensin-3 in pulmonary cells

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards <it>L. pneumophila</it>.</p> <p>Methods</p> <p>We investigated the effects of <it>L. pneumophila </it>and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis.</p> <p>Results</p> <p><it>L. pneumophila </it>induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on <it>L. pneumophila </it>replication.</p> <p>Conclusions</p> <p>Taken together, human pulmonary cells produce hBD-3 upon <it>L. pneumophila </it>infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.</p