Resorbable bioactive ceramic for treatment of bone infection.

Polymethylmethacrylate (PMMA) beads have been widely used in the treatment of bone infection over the last three decades. Although PMMA does offer a mechanism to quickly and effectively administer a localized dose of antibiotic to the site of infection, its efficacy is limited by its nonresorbability and nonbioactivity. Resorbable bioactive silica-calcium phosphate nanocomposite (SCPC75) was investigated as a novel controlled release carrier of vancomycin for the treatment of osteomyelitis. SCPC75 particles adsorbed significantly higher amount of vancomycin compared with PMMA. Moreover, SCPC75 provided a sustained release kinetics of therapeutic dose of vancomycin up to 35 days. The novel resorbable ceramic was able to release 95.5% of the adsorbed drug in an average dose of 12 microg/mL/day over 480 h (35 days). In conjunction with the sustained drug release, a controlled dissolution rate that led to 40% mass loss of SCPC75 was observed. On the other hand, PMMA provided a sustained release of a therapeutic dose of vancomycin for 14 days after which minimal concentration of the drug was detected. Moreover, PMMA retained 32% of the drug adsorbed onto its surface. The SCPC-vancomycin implant can serve a dual function: provide a sustained therapeutic dose of antibiotic to eradicate infection and stimulate bone cell differentiation and new bone formation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Activation of peripheral delta2 opioid receptors increases cardiac tolerance to ischemia/reperfusion injury Involvement of protein kinase C, NO-synthase, KATP channels and the autonomic nervous system.

AIMS: This study aims to investigate the role of peripheral delta(2) opioid receptors in cardiac tolerance to ischemia/reperfusion injury and to examine the contribution of PKC, TK, K(ATP) channels and the autonomic nervous system in delta(2) cardioprotection. MAIN METHODS: Deltorphin II and various inhibitors were administered in vivo prior to coronary artery occlusion and reperfusion in a rat model. The animals were monitored for the development of arrhythmias, infarct development and the effects of selected inhibitors. KEY FINDINGS: Pretreatment with peripheral and delta(2) specific opioid receptor (OR) antagonists completely abolished the cardioprotective effects of deltorphin II. In contrast, the selective delta(1) OR antagonist 7-benzylidenenaltrexone (BNTX) had no effect. The protein kinase C (PKC) inhibitor chelerythrine and the NO-synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester) also reversed both deltorphin II effects. The nonselective ATP-sensitive K+ (K(ATP)) channel inhibitor glibenclamide and the selective mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoic acid only abolished the infarct-sparing effect of deltorphin II. Inhibition of tyrosine kinase (TK) with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II but did not change its infarct-sparing action. SIGNIFICANCE: The cardioprotective mechanism of deltorphin II is mediated via stimulation of peripheral delta(2) opioid receptors. PKC and NOS are involved in both its infarct-sparing and antiarrhythmic effects. Infarct-sparing is dependent upon mitochondrial K(ATP) channel activation while the antiarrhythmic effect is dependent upon TK activation. Endogenous catecholamine depletion reduced antiarrhythmic effects but did not alter the infarct-sparing effect of deltorphin II.

Periodontal bacterial profiles in pregnant women: response to treatment and associations with birth outcomes in the obstetrics and periodontal therapy (OPT) study.

BACKGROUND: A recent clinical trial (Obstetrics and Periodontal Therapy [OPT] Study) demonstrated that periodontal therapy during pregnancy improved periodontal outcomes but failed to impact preterm birth. The present study evaluated seven target bacteria, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia (previously T. forsythensis), Prevotella intermedia, Campylobacter rectus, and Fusobacterium nucleatum, in subgingival dental plaque of pregnant women in the OPT Study and their association with birth outcomes. METHODS: Pregnant women were randomly assigned to receive periodontal treatment before 21 weeks' gestation or after delivery. Subgingival plaque was sampled at baseline (13 to 16 weeks; 6 days of gestation) and at 29 to 32 weeks. We analyzed subgingival plaque samples from women who experienced fetal loss, delivered a live preterm infant (preterm women), or delivered a full-term infant (full-term women). Samples were analyzed using quantitative polymerase chain reaction. Associations between preterm birth and bacterial counts and percentages were tested using multiple linear regression. RESULTS: No significant differences were observed at baseline between preterm and full-term women for any measured bacterial species or group of species, after accounting for multiple comparisons. Changes in bacterial counts and proportions during pregnancy also were not associated with birth outcomes. In full-term and preterm women, periodontal therapy significantly reduced (P <0.01) counts of all target species except for A. actinomycetemcomitans. CONCLUSIONS: In pregnant women with periodontitis, non-surgical periodontal therapy significantly reduced levels of periodontal pathogens. Baseline levels of selected periodontal pathogens or changes in these bacteria resulting from therapy were not associated with preterm birth.

Delta 2-specific opioid receptor agonist and hibernating woodchuck plasma fraction provide ischemic neuroprotection.

OBJECTIVES: The authors present evidence that the delta opioid receptor agonist Deltorphin-D(variant) (Delt-D(var)) and hibernating woodchuck plasma (HWP), but not summer-active woodchuck plasma (SAWP), can provide significant neuroprotection from focal ischemia in mice by a mechanism that relies in part on reducing nitric oxide (NO) release in ischemic tissue. METHODS: Cerebral ischemia was produced in wild-type and NO synthase-deficient (NOS(-/-)) mice by transient, 1-hour middle cerebral artery occlusion (MCAO). Behavioral deficits were determined at 22 hours and infarct volume was assessed at 24 hours after MCAO. Mice were treated with saline or Delt-D(var) at 2.0 and 4.0 mg/kg, or 200 microL of HWP or SAWP. NOS(-/-) mice were treated with either saline or Delt-D(var) at 4.0 mg/kg. NO release was determined using an N9 microglial cell line pretreated with delta- or mu-specific opioids and HWP or SAWP prior to activation with lipopolysaccharide and interferon-gamma. Nitrate in the medium was measured as an indicator of NO production. RESULTS: Infusion of Delt-D(var) or HWP (but not SAWP) decreased infarct volume and improved behavioral deficits following 1 hour of MCAO and 24 hours of reperfusion. In NOS(-/-) mice, endothelial NOS(+/+) is required to provide Delt-D(var)-induced neuroprotection. Delt-D(var) and HWP dose-dependently decreased NO release in cell culture, while SAWP and other delta- and mu-specific opioids did not. CONCLUSIONS: Delt-D(var) and HWP, but not SAWP, are effective neuroprotectant agents in a mouse model of transient MCAO. In cell culture, the mechanism of this ischemic neuroprotection may rely in part on their ability to block NO release.

Post-treatment with the novel deltorphin E, a delta2-opioid receptor agonist, increases recovery and survival after severe hemorrhagic shock in behaving rats.

Deltorphin E was investigated as a pharmaceutical intervention in the ischemic hemorrhagic model. To monitor the hemodynamic biomarkers mean arterial pressure (MAP) and heart rate (HR) and to facilitate i.v. injections, rats were surgically fitted with femoral artery and vein catheters under anesthesia. After removal of 48% of total blood volume (range, 12-15 mL), posthemorrhage i.v. injections of 5.5-mg/kg deltorphin E were found to significantly (P < 0.05) increase maximum MAP, pulse pressure, and survival after hemorrhage, whereas lactic acid concentration was decreased when compared with saline injections. The results for the 5.5-mg/kg deltorphin E-treated animals versus saline controls showed the following values (expressed as mean +/- SEM): maximum MAP, 58 +/- 7 vs. 35 +/- 9 mmHg, respectively; lactic acid, 6.5 +/- 1.25 vs. 8.9 +/- 0.12 mmol/L, respectively; pulse pressure, 47.9 +/- 0.55 vs. 38.3 +/- 0.44 mmHg, respectively; and at least a fourfold increase in survival, 331 +/- 18 vs. 50 +/- 8 min, respectively. Heart rate in deltorphin E-treated groups was not significantly different from that in saline-treated groups (maximum HR, 396 +/- 40 vs. 425 +/- 94 bpm, respectively). Using logistic analysis, deltorphin E did not significantly alter the baroreflex sensitivity. However, a significant deltorphin E dose-dependent correlation was found between survival time and lactic acid production. Increased pulse pressure was also correlated with survival. Glibenclamide, a potassium-sensitive adenosine triphosphate-sensitive channel blocker, did not interfere with the positive effects of deltorphin E. Only the antagonists tested, known to affect delta(2)-opioid receptors, interfered with the deltorphin E survival benefit after hemorrhage. As a conclusion, deltorphin E is an effective pharmaceutical intervention in severe hemorrhagic shock and, perhaps, in other ischemic shock scenarios when administered after the onset of stress. Therefore, deltorphin E may have clinical potential.

24-hour pretreatment with delta opioid enhances survival from hemorrhagic shock.

OBJECTIVES: Delta opioids have been shown to confer ischemic preconditioning and pharmacologic ischemic preconditioning to the myocardium. However, their role in providing extended pharmacologic ischemic preconditioning in hemorrhagic shock has not been explored. The authors examined the effects of 24-hour preinfusions of a selective delta opioid receptor agonist, Deltorphin-Dvariant (Delt-Dvar), on hemodynamic stability and duration of survival in a rat model of severe hemorrhagic shock. METHODS: Conscious Sprague-Dawley rats with indwelling catheters were hemorrhaged at a rate of 3.18 mL/l00 g over 20 minutes. Twenty-four hours before hemorrhage, the control group (n = 14) was infused with 1.0 mL lactated Ringer's solution, and the Delt-Dvar-treated group (n = 22) was infused with 5.0 mg/kg Delt-Dvar in 1.0 mL lactated Ringer's solution. Rats were continuously monitored for heart rate (HR), mean arterial pressure, and four-hour survival rates. Plasma lactate levels were determined at the beginning of hemorrhage and the end of hemorrhage. RESULTS: At 240 minutes, only one of 14 controls (7.1%) survived, while 16 (72.7%) of the 22 experimental rats survived. No significant differences in heart rate between controls and Delt-Dvar-treated rats were noted. Increases in mean arterial pressure of Delt-Dvar-treated rats at the beginning of hemorrhage and at the end of hemorrhage were found to be significant (p < 0.05). At 240 minutes, heart rate and mean arterial pressure were not different between the single surviving control and the Delt-Dvar group. At the end of hemorrhage, lactate levels in the Delt-Dvar-treated group were 8.5 (+/- 0.5) mmol/L versus 10.8 (+/- 0.6) mmol/L (p < 0.05) in the control group. CONCLUSIONS: Twenty-four-hour pretreatment with Delt-Dvar decreases plasma lactate levels and improves hemodynamic stability and survival during hemorrhagic shock. The use of delta-specific opioids may improve survival from hemorrhagic shock and have clinical utility in providing ischemic protection in scenarios of planned ischemia.

A delta2-opioid agonist inhibits p38 MAPK and suppresses activation of murine macrophages.

BACKGROUND: delta-Opioid agonists have been shown to attenuate ischemic organ injury in multiple models. The purpose of the present study was to determine if delta-opioid agonists could inhibit proinflammatory cytokine production by macrophages. MATERIAL AND METHODS: Murine macrophages (RAW 264.7) were pretreated for 4 h with media, a dose range (10(-4) to 10(-7) M) of DADLE ([D-Ala2], D-Leu5]-enkephalin, a nonspecific delta-opioid receptor agonist), a dose range (10(-4) to 10(-7) M) of DPDPE ([D2,5Pen]-enkephalin, a specific delta1-opioid receptor agonist), or a dose range (10(-4) to 10(-7) M) of Deltorphin-Dvariant (a specific delta2 opioid receptor agonist) and then incubated with 0.1 microg/ml lipopolysaccharide (LPS) for 1 or 4 h. Cytokine levels were measured by enzyme-linked immunosorbent assay. Activation of NF-kappaB, AP-1, and p38 MAPK were determined by mobility shift assays and Western blot. RESULTS: LPS induced significant increases in TNFalpha and MIP-2 production. Deltorphin-Dvariant, but not DADLE or DPDPE, dose-dependently reduced both TNFalpha and MIP-2 production. Deltorphin-Dvariant did not alter activation of the transcription factors NF-kappaB or AP-1, but greatly reduced activation of p38 MAPK. CONCLUSIONS: The data show that delta2- but not delta1-opioid agonists suppress LPS-induced p38 MAPK activation and expression of TNFalpha and MIP-2.

Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein.

The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid beta-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k(cat), with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.