Differentiation of osteoprogenitor cells is affected by trauma-haemorrhage.

INTRODUCTION: In multiple trauma patients an increased incidence of delayed healing and non-union has been observed. The exact mechanisms underlying this delayed fracture healing are still not fully understood. MATERIAL AND METHODS: 40 male C57BL/6N-mice underwent standardized midline laparotomy and pressure-controlled haemorrhage (TH) or implantation of catheters without blood loss (sham procedure). Animals were sacrificed 24h or 72 h later. Osteoprogenitor cells derived from bone marrow were isolated and differentiated into osteoblasts for 20 days and osteoclasts for 7 days. Osteoblast mineralization and osteoclast numbers were determined, and gene expression of Alpl, Bglap, Opg, Rankl was measured in osteoblast cell culture, as well as gene expression of Rank, Ctsk and Nfatc1 was determined in osteoclast cell culture. Furthermore, plasma Opg, Rankl and TRAP were measured. RESULTS: Mineralization capacity of osteoblasts was unchanged after TH, but Alpl gene expression after 23 days was significantly decreased compared to sham. Osteoclast number of group TH 8 days was significantly decreased compared to sham. Furthermore, gene expression of Ctsk and Nfatc1 were increased in group TH 10 days compared to group TH 8 days. Plasma Opg concentration was significantly elevated and Rankl concentrations were significantly declined in TH groups compared to sham groups after 24h and 72 h. CONCLUSION: TH results in a diminished osteoclast number after 8 days, whereas differentiation of osteoblasts seems to be unaffected. The reduction of osteoclast number seems to be mediated through the Rankl-Opg-signalling pathway. However, further studies in models including a fractured extremity with a longer observation period are needed to identify the relevance of the Rankl-Opg- pathway in delayed fracture healing after TH and to focus on possible therapeutic interventions.

Depletion of NK cells in a murine polytrauma model is associated with improved outcome and a modulation of the inflammatory response.

Sepsis and associated diseases such as systemic inflammatory response syndrome and multiple organ dysfunction syndrome represent common posttraumatic complications on intensive care units induced by a variety of body defense mechanisms. Natural killer (NK) cells are part of the innate immune system. They are thought to play an important role in the development of such syndromes by interplay with other immune cell types and subsequent activation of the inflammatory cascade. To test this hypothesis, NK cells were depleted by administration of antimouse asialo-GM1 antibody in a murine polytrauma model consisting of femur fracture, hemorrhagic shock, and subsequent sepsis. Mortality and immune parameters such as cytokine expression in lung and liver, lymphocyte phenotyping, lymphocyte apoptosis, and organ pathology were determined 96 h after sepsis induction. Survival values showed 50% in the control sepsis group and 100% after NK cell depletion. Thus, NK cell depletion resulted in 50% mortality reduction. Furthermore, we found reductions in the inflammatory response, represented by IL-6 expression in liver, and a decrease in infiltrating neutrophils in the liver and lung. In addition, lymphocyte apoptosis in spleen was decreased by depletion of NK cells. Taken together, these data demonstrate that NK cells contribute to the pathogenetic pathways in a murine polytrauma model. One main mechanism of action seems to be the induction of systemic inflammatory events. Thus, depletion of NK cells results in attenuated inflammation and an overall improvement in outcome. Therefore, NK cells can be considered as important targets for therapeutic strategies.

Dehydroepiandrosterone administration modulates endothelial and neutrophil adhesion molecule expression in vitro.

INTRODUCTION: The steroid hormone dehydroepiandrosterone (DHEA) exerts protecting effects in the treatment of traumatic and septic complications in several animal models. This effect goes along with reduced amounts of infiltrating immune cells in organs such as lung and liver. However, the underlying mechanisms of DHEA action are still not known. Adhesion molecules are important for the extravasation of neutrophils into organs where they may exhibit detrimental effects. Therefore, we investigated the in vitro effect of DHEA on the expression pattern of adhesion molecules of human endothelial cells and neutrophils. METHODS: Endothelial cells derived from human umbilical cord were subjected to an lipopolysaccharide (LPS) challenge. DHEA was administered in two different concentrations, 10(-5) M and 10(-8) M, as a single stimulus or in combination with LPS challenge. After two, four and 24 hours, fluorescence activated cell sorter (FACS) analysis for vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin was performed. Neutrophils were freshly isolated from blood of 10 male healthy volunteers, stimulated the same way as endothelial cells and analyzed for surface expression of L-selectin, CD11b and CD18. RESULTS: In the present study, we were able to demonstrate effects of DHEA on the expression of every adhesion molecule investigated. DHEA exhibits opposite effects to those seen upon LPS exposure. Furthermore, these effects are both time and concentration dependent as most DHEA specific effects could be detected in the physiological concentration of 10(-8) M. CONCLUSION: Thus, we conclude that one mechanism by which DHEA may exert its protection in animal models is via the differential regulation of adhesion molecule expression.

Optional deductibles in social health insurance systems: findings from Germany.

A fundamental aspect of the German health insurance system is the principle of solidarity. At the same time, it is possible for certain socio-economic groups to opt out of the otherwise compulsory system. To determine whether rates incorporating deductibles are compatible with the principles of solidarity and have the ability to heighten the appeal of statutory health insurance (SHI) funds compared with private health insurance companies, Germany's third largest SHI fund, Techniker Krankenkasse, implemented a pilot scheme involving the use of deductibles. Preliminary scientific evaluations of the pilot scheme indicate three main results for these deductibles: Firstly, they are compatible with the principles of solidarity in the statutory health insurance system; secondly, they provide an effective means of preventing defection to private health insurance companies and thirdly, they reduced the volume of insurance claims (moral hazard).

Role of adhesion molecule ICAM in the pathogenesis of polymicrobial sepsis.

INTRODUCTION: Intercellular adhesion molecule-1 (ICAM-1) is thought to be involved in polymorphonuclear leukocytes (PMNL) recruitment and secondary organ damage in response to infection and inflammation. The precise role of ICAM-1 in disease progression is unknown and remains a topic of controversy. The aim of this study was to investigate the effect of ICAM-1 on histological changes and cytokine synthesis in a murine model of polymicrobial sepsis. METHODS: Polymicrobial sepsis was induced in experimental animals by caecal ligation and puncture (CLP). A control group was formed using sham laparotomy without CLP. In order to ascertain the role of ICAM-1 in the response, procedures were performed in both ICAM-1 knockout animals (ICAM-1-/-) and in C57BL/6 mice that were not genetically modified (wild type, WT). Clinical response was observed daily, morphological changes occurring in the lung and liver were studied using light microscopy and quantified using a scoring system. Plasma concentrations of various cytokines (TNF-alpha, IL-6, IL-10) were measured via ELISA. RESULTS: In ICAM-1-/- mice a less severe clinical response to induced sepsis was observed with significantly less weight loss and hypothermia. A significantly lower mortality rate was observed in ICAM-1-/- mice (12.5% vs. WT: 45.5%) and no significant histological changes were apparent in pulmonary or hepatic tissue on light microscopy following CLP. In WT animals however, significant evidence of leukocyte infiltration and interstitial thickening in pulmonary tissue was observed. Similarly, hepatic tissue sinusoidal widening and hydropic degeneration was present. In addition, pro- and anti-inflammatory cytokine synthesis in ICAM-1-/- animals was significantly attenuated when compared to WT mice. (ICAM-1-/-: TNF-alpha: 67.7+/-12.1pg/microl; IL-6: 208.9+/-26.7pg/microl; IL-10: 34.6+/-5.8pg/microl; WT: TNF-alpha: 840.7+/-150.2pg/microl; IL-6: 3100.2+/-1052.3 pg/microl; IL-10: 1550.1+/-495.7 pg/microl). DISCUSSION: This study suggests that ICAM-1 has an important pathophysiological role in the response to polymicrobial sepsis. It would appear that absence of this molecule impairs the ability of PMNL to migrate into organ tissues and reduces consequent secondary organ damage resulting in improved clinical status and overall survival. Further investigation into the effectiveness of ICAM-1 modulation in the treatment of sepsis is warranted.

Inhibition of nucleoside transport by new analogues of 4-nitrobenzylthioinosine: replacement of the ribose moiety by substituted benzyl groups.

4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. However, its highly polar nature is unfavorable for oral absorption and/or penetration into the CNS. In the search for compounds with lower polarity than NBTI we replaced its ribose moiety by substituted benzyl groups. Halogen, hydroxyl, (trifluoro)methyl(-oxy), nitro, and amine functionalities were among the substituents at the benzyl group. In general, substitution of the benzyl group resulted in a lower affinity for ENT1. Only 2-hydroxyl substitution showed a higher affinity. Most likely this is the result of hydrogen bonding. Substitution at the 2-position of the benzyl group with aryl groups was also addressed. Compared to parent compound carrying a 2-phenylbenzyl group, all synthesized analogues gave higher affinities. Introduction of fluoro, trifluoromethyl, methoxy, and hydroxyl groups at the phenyl group clearly showed that addition to the 4-position was preferable. Despite the highly different character of a ribose and a benzyl group, Ki values in the low nanomolar range were obtained for the benzyl-substituted derivatives. Compound 35, LUF5919, and compound 60, LUF5929, displayed the highest affinity (Ki = 39 nM for both compounds), having a polar surface area of 101 A2 and 85 A2, respectively.