In contrast to endogenous opioids, the highly addictive drug morphine activates the -opioid receptor without causing its rapid endocytosis. It has recently been reported that coapplication of low concentrations of [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) facilitates the ability of morphine to stimulate -opioid receptor endocytosis and prevents the development of morphine tolerance in rats. To investigate the clinical relevance of this finding for analgesic therapy, the endocytotic efficacies of a series of clinically used opioids were determined, and the effect of a combination of these drugs with morphine on the -opioid receptor endocytosis in receptor-expressing human embryonic kidney (HEK) 293 cells was quantified. The combination of morphine and opioid drugs with high endocytotic efficacies (e.g., DAMGO, etonitazene, sufentanil, ␤-endorphin, piritramide, or methadone) did not result in a facilitation of morphine-mediated endocytosis but rather in a decrease of the receptor endocytosis mediated by the tested opioid drugs. These findings demonstrate a partial agonistic effect of morphine on the agonist-induced receptor endocytosis. Moreover, we demonstrated that the endocytotic potencies of opioid drugs are negatively correlated with their ability to cause receptor desensitization and opioid tolerance in HEK 293 cells. These results strongly support the hypothesis that -opioid receptor endocytosis counteracts receptor desensitization and opioid tolerance by inducing fast receptor reactivation and recycling. In addition, it is shown that agonist-induced receptor endocytosis facilitates the compensatory up-regulation of the cAMP pathway, a cellular hallmark of opioid withdrawal. Our findings suggest that opioids with high endocytotic efficacies might cause reduced opioid tolerance but can facilitate compensatory mechanisms, resulting in an enhanced opioid dependence.In vitro and in vivo studies revealed that on the cellular level, long-term opioid treatment leads to a rapid receptor desensitization with reduction of agonist response followed by internalization of receptors. An important mechanism of -opioid receptor (MOR) desensitization is the phosphorylation of intracellular receptor domains by G protein-coupled receptor kinases or second messenger-regulated protein kinases, such as Ca 2ϩ /calmodulin-dependent protein kinase II

Antimicrobial peptides (APs) are an important part of the innate immune system in epithelial and non-epithelial surfaces. So far, many different antimicrobial peptides from various families have been discovered in non-vertebrates and vertebrates. They are characterized by antibiotic, antifungal and antiviral activities against a variety of microorganisms. In addition to their role as endogenous antimicrobials, APs participate in multiple aspects of immunity. They are involved in septic and non-septic inflammation, wound repair, angiogenesis, regulation of the adaptive immune system and in maintaining homeostasis. Due to those characteristics AP could play an important role in many practical applications. Limited therapeutic efficiency of current antimicrobial agents and the emerging resistance of pathogens require alternate antimicrobial drugs. The purpose of this review is to highlight recent literature on functions and mechanisms of APs. It also shows their current practical applications as peptide therapeutics and bioactive polymers and discusses the possibilities of future clinical developments.

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.