A short artificial antimicrobial peptide shows potential to prevent or treat bone infections.

Infection of bone is a severe complication due to the variety of bacteria causing it, their resistance against classical antibiotics, the formation of a biofilm and the difficulty to eradicate it. Antimicrobial peptides (AMPs) are naturally occurring peptides and promising candidates for treatment of joint infections. This study aimed to analyze the effect of short artificial peptides derived from an optimized library regarding (1) antimicrobial effect on different bacterial species, (2) efficacy on biofilms, and (3) effect on osteoblast­like cells. Culturing the AMP-modifications with Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus (including clinical isolates of MRSA and MSSA) and Staphylococcus epidermidis identified one candidate that was most effective against all bacteria. This AMP was also able to reduce biofilm as demonstrated by FISH and microcalorimetry. Osteoblast viability and differentiation were not negatively affected by the AMP. A cation concentration comparable to that physiologically occurring in blood had almost no negative effect on AMP activity and even with 10% serum bacterial growth was inhibited. Bacteria internalized into osteoblasts were reduced by the AMP. Taken together the results demonstrate a high antimicrobial activity of the AMP even against bacteria incorporated in a biofilm or internalized into cells without harming human osteoblasts.

In vivo quantification of gentamicin released from an implant coating.

Drug-releasing implants are gaining increasing interest. The present study reports a detailed physicochemical analysis of a polymeric coating based on poly(D,L-lactide) and the incorporated gentamicin combined with an in vitro and in vivo study of the gentamicin release. Differential scanning calorimeter, Fourier transform infrared spectroscopy, gel permeation chromatography and high-performance liquid chromatography showed no effect of the gamma sterilisation on the coating components or an interaction of the polymer and the gentamicin. Microbiological analysis revealed an inhibition of bacterial growth on the implant surface. For the in vivo study, gentamicin-coated wires were implanted into the tibiae of rats and harvested at different time points up to day 42. To monitor the release in vivo, gentamicin was quantified in serum, bone, endosteum, kidney, and on the explanted wires. Gentamicin was detectable over a time period of 42 days in the endosteum, up to seven days in the kidney, up to 4 h in the bone and at the end of the experiment on one of eight wires. The locally released gentamicin caused no histological changes of the kidney. Microbiologically active concentrations of released gentamicin were found in the endosteum up to 4 h after implantation. The combination of different methods supports the individual results, where quantification is complemented by visualisation or antimicrobial activity. This work demonstrates that the coating procedure results in no substantial alteration of the incorporated drug and that the in vitro burst release occurs also in vivo.

Analysis of parameters influencing the release of antibiotics mixed with bone grafting material using a reliable mixing procedure.

Local infections arising from fracture fixation, defect reconstruction or joint replacement can cause extreme pain and impaired healing, lead to revision operations, prolong hospital stay and increase costs. Treatment options including prophylaxis are afforded by the use of grafts and biomaterials loaded with antibiotics. These can produce local therapeutic concentrations with a reduced systemic concentration and reduced systemic side-effects. Patient-specific loading of osteogenic graft materials with antibiotic could be an important option for orthopaedic surgeons. A local therapeutic concentration must be available for the desired duration and cytotoxic effects must be kept within an acceptable range. The present study investigates a simple and reliable mixing procedure that could be used for the perioperative combination of antibiotic powders and solutions with bone grafting materials. The potential influence of concentration and sampling regime on the release kinetics of gentamicin, tobramycin and vancomycin was studied over a period of 56days and potency and cytotoxicity were evaluated. In all treatment groups, gentamicin and tobramycin were completely released within 3days whilst vancomycin was released over a period of 14days. The results clearly show that the main parameter influencing release is the molecular weight of the drug. Growth of Staphylococcus aureus was inhibited in all 3 treatment groups for at least 3days. Cell viability and alkaline phosphatase activity of primary osteoblast-like cells were not significantly affected by the antibiotic concentrations obtained from the elution experiments. Bone grafting is an established component of surgery for bone defect filling and for biological stimulation of healing. Patient-specific enhancement of such procedures by incorporation of antibiotics for infection prevention or by addition of cytokines for promotion of impaired healing or for treatment of critical size defects will be a relevant issue in the future.

In vitro testing of the osteoinductive potential of different bony allograft preparations.

INTRODUCTION: Bony allografts are used frequently in the clinic for bone defect filling, however, less comparative data concerning their osteoinductive potential are available. AIM: The purpose of the present study was the comparative analysis of different allograft preparations. From five donors, we investigated fresh-frozen cancellous bone (native), peracetic acid­ethanol sterilized (PES) cancellous bone, cortical bone and demineralised bone matrix (DBM). In addition, two commercially available DBM products from five different donors were analyzed: Allomatrix® (Wright Medical Technology Inc.) and DBX putty® (Synthes GmbH). For positive control and as a clinically used growth factor, BMP-2 was chosen. METHOD: To investigate the osteoinductivity C2C12 cells were cultured with the different materials and the effect on cell proliferation and alkaline phosphatase activity were measured. RESULT: Proliferation was significantly enhanced by the native cancellous bone, Allomatrix, and BMP-2 and decreased by the PES-processed cancellous bone. The osteogenic differentiation was significantly enhanced by BMP-2 and the two commercial DBM products and decreased by PES-sterilized cancellous bone. All tested materials revealed a high donor-dependent variability. This is the first comparative study on the osteoinductivity of bony allografts frequently used in clinic.

GLANCE: results of a phase 2, randomized, double-blind, placebo-controlled study.

OBJECTIVE: To evaluate the safety and tolerability of natalizumab when added to glatiramer acetate (GA) in patients with relapsing multiple sclerosis. The primary outcome assessed whether this combination would increase the rate of development of new active lesions on cranial MRI scans vs GA alone. METHODS: This phase 2, randomized, double-blind, placebo-controlled study included patients aged 19 to 55 years who were treated with GA for at least 1 year before randomization and experienced at least one relapse during the previous year. Patients received IV natalizumab 300 mg (n = 55) or placebo (n = 55) once every 4 weeks plus GA 20 mg subcutaneously once daily for < or = 20 weeks. RESULTS: The mean rate of development of new active lesions was 0.03 with combination therapy vs 0.11 with GA alone (p = 0.031). Combination therapy resulted in lower mean numbers of new gadolinium-enhancing lesions (0.6 vs 2.3 for GA alone, p = 0.020) and new/newly enlarging T2-hyperintense lesions (0.5 vs 1.3, p = 0.029). The incidence of infection and infusion reactions was similar in both groups; no hypersensitivity reactions were observed. One serious adverse event occurred with combination therapy (elective hip surgery). With the exception of an increase in anti-natalizumab antibodies with combination therapy, laboratory data were consistent with previous clinical studies of natalizumab alone. CONCLUSION: The combination of natalizumab and glatiramer acetate seemed safe and well tolerated during 6 months of therapy.

Extinction of ethanol-induced conditioned place preference and conditioned place aversion: effects of naloxone.

Four experiments examined the effect of naloxone pretreatment on the expression and extinction of ethanol-induced conditioned place preference (experiments 1, 2, 4) or conditioned place aversion (experiments 1, 3). DBA/2 J mice received four pairings of a distinctive tactile (floor) stimulus (CS) with injection of ethanol (2 g/kg) given either immediately before or after 5-min exposure to the CS. A different stimulus was paired with injection of saline. Pre-CS injection of ethanol produced conditioned place preference, whereas post-CS injection of ethanol produced conditioned place aversion. Both behaviors extinguished partially during repeated choice testing after vehicle injection. Naloxone (10 mg/kg) had little effect on the initial expression of conditioned place preference, but facilitated its extinction. Moreover, repeated naloxone testing resulted in the expression of a weak conditioned place aversion to the CS that initially elicited a place preference. In contrast, naloxone (1.5 or 10 mg/kg) enhanced expression of conditioned place aversion, thereby increasing its resistance to extinction. A control experiment (experiment 4) indicated that repeated testing with a different aversive drug, lithium chloride, did not affect rate of extinction or produce an aversion to the CS previously paired with ethanol. These findings do not support the suggestion that naloxone facilitates the general processes that underlie extinction of associative learning. Also, these data are not readily explained by the conditioning of place aversion at the time of testing. Rather, naloxone's effects appear to reflect a selective influence on maintenance of ethanol's conditioned rewarding effect, an effect that may be mediated by release of endogenous opioids. Overall, these findings encourage further consideration of the use of opiate antagonists in the treatment of alcoholism.

Ethanol-induced conditioned place aversion in rats: effect of interstimulus interval.

The purpose of this study was to examine the effect of interstimulus interval (ISI) on ethanol-induced place aversion in rats. Six groups of rats initially received four pairings of a distinctive floor stimulus (CS+) with ethanol (1 g/kg, I.P.) and four pairings of a different floor stimulus (CS-) with saline. Groups -30, -15, -10, -5, 0, and 5 were injected 30, 15, 10, 5, or 0 min before, or 5 min after exposure to the 5 min CS, respectively. After testing for place aversion, all groups were exposed to an additional set of conditioning trials using a higher dose of ethanol (1.5 g/kg). During the first test, only groups 0 and -15 exhibited conditioned place aversion. However, during the second test, all groups showed conditioned aversion except group -30. The results suggest that ethanol's aversive effects dissipate by 30 min postinjection or that it is more difficult to associate those effects with short-duration external stimuli at long backward intervals. In contrast to recent findings with mice, the direction of ethanol-induced place conditioning was not altered in rats exposed to different ISIs.

Transcriptional analysis of mga, a regulatory gene in Streptococcus pyogenes: identification of monocistronic and bicistronic transcripts that phase vary.

Transcription of several surface virulence proteins of Streptococcus pyogenes is regulated by Mga, a protein that shows homology to response regulators of two-component signal-transducing systems. Two of these surface virulence proteins, M protein and C5a peptidase, undergo phase variation. To determine whether Mga itself undergoes phase variation and might allow the phasing switch to coordinate the activity of these genes, expression of the mga gene was analyzed. We show for the first time that there are two mga-specific transcripts: a 3.8-kb bicistronic message that includes both mga and emm12 genes and a monocistronic 1.6-kb mga message. Both transcripts phase vary and are present in higher amounts in M+ variants than in M- variants. Incubation of RNA with rifampicin indicates that the smaller 1.6-kb message is not a processed product. Two promoters were mapped upstream of mga: P1 at position 666 (-395) and P2 at position 978 (-83). In strain CS46 (delta mga), transcription initiation from the P1 promoter does not occur, and multiple start sites are found around the P2 promoter. Complementation experiments indicate that sequences upstream of the P2 promoter are required for activation of emm12 and scpA by Mga in trans.

The effects of naloxone on expression and acquisition of ethanol place conditioning in rats.

Naloxone has been shown to facilitate extinction of ethanol-induced conditioned place preference (CPP) in mice. The present-study extended these findings by examining naloxone's effect on the expression (Experiment 1) and acquisition (Experiment 2) of place conditioning with ethanol in rats. In Experiment 1, after place conditioning with ethanol (1.8 g/kg, I.P.), groups N0, N1.5, and N10 received 0, 1.5, or 10 mg/kg naloxone before testing. As expected, ethanol produced a robust conditioned place aversion (CPA). However, naloxone had no effect on expression of CPA. In contrast to studies with mice, the endogenous opioid system does not appear to be involved in the conditioned motivational effects of ethanol in rats. In Experiment 2, groups SE1 and SE2, NS(1.5), NE(1.5), and NE(10), received ethanol alone (1.2 g/kg), naloxone alone (1.5 mg/kg), naloxone 1.5 mg/kg plus ethanol, and naloxone 10 mg/kg plus ethanol during acquisition, respectively. All naloxone-treated groups exhibited CPA. Moreover, group NE(1.5) showed a stronger CPA than group NS(1.5). The CPA produced by coadministration of naloxone and ethanol was attributed to naloxone's effects on the neural processes underlying ethanol's unconditioned aversive effects, or to other nonspecific effects on ethanol's motivational properties.

Reduced sensitivity to ethanol reward, but not ethanol aversion, in mice lacking 5-HT1B receptors.

Various serotonergic receptor systems are thought to influence the motivational effects of ethanol. This experiment characterized the acquisition of ethanol-induced conditioned taste aversion and ethanol-induced conditioned place reference in mutant knockout mice lacking 5-HT1b receptors. In the taste conditioning procedure, adult homozygous knockout mice (-/-) and homozygous wild-type mice (+/+) received access to 0.2 M NaCl solution, followed immediately by intraperitoneal injection of 0 to 4 g/kg of ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. In the place conditioning procedure, knockout and wild-type mice received six pairings of a tactile stimulus with ethanol (2 g/kg, i.p.). A different tactile stimulus was paired with saline. Ethanol produced increases in locomotor activity, with wild-type mice showing higher levels of ethanol-stimulated activity than knockout mice during conditioning trials 5 and 6. Wild-type mice demonstrated conditioned place preference for the ethanol-paired stimulus. In contrast, knockout mice showed no evidence of place conditioning. These results are generally consistent with an important role for serotonergic systems in ethanol reward and specifically indicate that 5-HT1b receptors are important for ethanol's rewarding effects but not for ethanol's aversive effects.

The relative salience of morphine and contextual cues as conditioned stimuli.

Two experiments were conducted to delineate further the properties of conditioning when morphine is used as a conditioned stimulus (CS) in the conditioned suppression of drinking paradigm. Experiment 1 used a test for overshadowing designed to compare the relative salience of contextual cues (metal box) and morphine induced cues (6 mg/kg, IP) as CSs when each was paired with a foot shock unconditioned stimulus (US) in water deprived rats. Six groups (six rats each) were exposed to conditioning procedures during which the conditioning context was present 19 h (groups 1 and 2), 90 min (groups 3 and 4), or 5 min (groups 5 and 6) before shock onset, and morphine (in groups 1, 3, and 5) or saline (in groups 2, 4, and 6) was injected 10 min before shock. Subsequently, the magnitude of suppression of drinking in response to morphine, to the metal box, and to morphine plus the metal box was measured. Only group 1 (19 h group) suppressed drinking in response to morphine, while groups 3-6 suppressed drinking whenever tested in the metal box. The results indicate that morphine cues acted as a CS that elicited suppression of drinking in group 1, and that contextual cues present up to 90 min before morphine cues overshadowed morphine. Experiment 2 showed that expression of the conditioned response to morphine was blocked by naloxone.

VirR and Mry are homologous trans-acting regulators of M protein and C5a peptidase expression in group A streptococci.

Transcription of the group A streptococcal M12 protein gene (emm12) and the C5a peptidase gene (scpA), which encodes an inhibitor of complement-mediated chemotaxis, was previously shown to depend on a third genetic locus, designated virR. A 1.6 kb region of DNA which is 200 bp upstream of emm12 and is thought to contain the virR locus, was sequenced. An open reading frame which overlaps deletion mutations that define virR was identified. The sequence of the encoded VirR protein, which was deduced to contain 499 amino acids, is characteristic of cytoplasmic proteins. Comparison of the VirR protein to a variety of DNA binding proteins, such as lambda Cro, revealed a DNA binding motif. VirR was also compared to the M6 positive regulator, mry, and found to be 98% homologous. The predicted virR promoter is preceded by two sets of inverted repeats, in contrast to mry which is preceded by one repeat. Introduction of virR on the shuttle vector pAM401 into a strain of group A Streptococcus with a deletion in the chromosomal virR gene demonstrated that the VirR protein activated transcription of both emm12 and scpA genes in trans. Analysis of RNA by Northern blot using virR-specific probes identified two virR transcripts, a 1.6 kb transcript which corresponds to the predicted size of the gene, and a second transcript, 3.5 kb, which also overlaps virR. These results demonstrate that virR and mry are structurally and functionally very similar and show that the former is a trans activator of both M protein and C5a peptidase synthesis.

Morphine as a conditioned stimulus in a conditioned emotional response paradigm.

A Pavlovian conditioning experiment was conducted to determine whether morphine (6 mg/kg, IP) could act as a conditioned stimulus (CS) when paired with an electric shock unconditioned stimulus (US), and later produce a conditioned suppression of drinking (CR) in water deprived rats. Seven groups were tested for conditioning after exposure to one of the following conditioning procedures: (1) morphine paired with shock; (2) morphine alone with no shock; (3) shock but no morphine; (4) no shock and no morphine; (5) morphine paired with vocalizations of shocked rats; (6) saline paired with shock; (7) saline alone with no shock. Groups 1 and 2 tested whether morphine could act as a CS. Groups 3 and 4 tested for sensitization. Group 5 tested whether exposure to the vocalizations of other rats could act as a US when paired with a morphine CS. Groups 6 and 7 tested whether cues associated with the injection procedure could act as a CS. Only subjects in group 1 showed conditioned suppression of drinking, when compared to control groups. Overall, the results indicate that morphine could act as a conditioned stimulus and that several of the more obvious possible sources of artifact did not significantly contribute to the CR that is produced.

A unique enhancement of associative learning produced by methylenedioxyamphetamine.

The effects of methylenedioxyamphetamine (MDA) on classical conditioning of the rabbit's nictitating membrane response were assessed in four experiments. Experiment 1 established a dose-effect curve for MDA doses of 0, 1, 3, and 10µmol/kg. Both the 3 and 10µmol/kg doses significantly enhanced the rate of learning. Experiment 2 established that the 10µmol/kg dose of MDA had no effect on non-associative determinants of responding to the conditioned stimulus (CS). However, this dose of MDA apparently sensitized the response to the unconditioned stimulus (US). Experiments 3 and 4 assessed the effects of the 10µmol/kg dose of MDA on sensory processing of the CS and US, respectively. MDA had no effect on the intensity thresholds for eliciting responses to either of these stimuli. Thus, the enhanced rate of acquisition observed in experiment 1 cannot be attributed to an increase in nonassociative responding to the CS or to enhanced sensory processing of the CS or US. MDA apparently enhanced the rate of acquisition in experiment 1 by facilitating the association between the CS and US. These findings are unique in that no other hallucinogen we have examined has enhanced acquisition without also affecting either non-associative responding or sensory processing of the CS and US.