Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

Biomimetic in vitro oxidation of lapachol: A model to predict and analyse the in vivo phase I metabolism of bioactive compounds

The bioactive naphtoquinone lapachol was studied in vitro by a biomimetic model with Jacobsen catalyst (manganese(III) salen) and iodosylbenzene as oxidizing agent. Eleven oxidation derivatives were thus identified and two competitive oxidation pathways postulated. Similar to Mn(III) porphyrins, Jacobsen catalyst mainly induced the formation of para-naphtoquinone derivatives of lapachol, but also of two ortho-derivatives. The oxidation products were used to develop a GC MS (SIM mode) method for the identification of potential phase I metabolites in vivo. Plasma analysis of Wistar rats orally administered with lapachol revealed two metabolites, alpha-lapachone and dehydro-alpha-lapachone. Hence, the biomimetic model with a manganese salen complex has evidenced its use as a valuable tool to predict and elucidate the in vivo phase I metabolism of lapachol and possibly also of other bioactive natural compounds. (C) 2012 Elsevier Masson SAS. All rights reserved.

A phase I clinical trial of a new 5-valent rotavirus vaccine

We conducted a phase I, double-blind, placebo-controlled trial to evaluate a new 5-valent oral rotavirus vaccine’s safety and immunogenicity profiles. Subjects were randomly assigned to receive 3 orally administered doses of a live-attenuated human-bovine (UK) reassortant rotavirus vaccine, containing five viral antigens (G1, G2, G3, G4 and G9), or a placebo. The frequency and severity of adverse events were assessed. Immunogenicity was evaluated by the titers of anti-rotavirus IgA and the presence of neutralizing antibodies anti-rotavirus. No severe adverse events were observed. There was no difference in the frequency of mild adverse events between experimental and control groups. The proportion of seroconversion was consistently higher in the vaccine group, for all serotypes, after each one of the doses. The 5-valent vaccine has shown a good profile of safety and immunogenicity in this small sample of adult volunteers.

Acetylcysteine for Prevention of Renal Outcomes in Patients Undergoing Coronary and Peripheral Vascular Angiography Main Results From the Randomized Acetylcysteine for Contrast-Induced Nephropathy Trial (ACT)

Background-It remains uncertain whether acetylcysteine prevents contrast-induced acute kidney injury. Methods and Results-We randomly assigned 2308 patients undergoing an intravascular angiographic procedure with at least 1 risk factor for contrast-induced acute kidney injury (age >70 years, renal failure, diabetes mellitus, heart failure, or hypotension) to acetylcysteine 1200 mg or placebo. The study drugs were administered orally twice daily for 2 doses before and 2 doses after the procedure. The allocation was concealed (central Web-based randomization). All analysis followed the intention-to-treat principle. The incidence of contrast-induced acute kidney injury (primary end point) was 12.7% in the acetylcysteine group and 12.7% in the control group (relative risk, 1.00; 95% confidence interval, 0.81 to 1.25; P = 0.97). A combined end point of mortality or need for dialysis at 30 days was also similar in both groups (2.2% and 2.3%, respectively; hazard ratio, 0.97; 95% confidence interval, 0.56 to 1.69; P = 0.92). Consistent effects were observed in all subgroups analyzed, including those with renal impairment. Conclusions-In this large randomized trial, we found that acetylcysteine does not reduce the risk of contrast-induced acute kidney injury or other clinically relevant outcomes in at-risk patients undergoing coronary and peripheral vascular angiography.

The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K+ channels. Crotalphine (0.2 or 5 mu g/kg, orally; 0.0006 mu g/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of delta-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of kappa-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K+ channel blocker. The results suggest that peripheral delta-opioid and kappa-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain. Behavioural Pharmacology 23:14-24 (C) 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. (C) 2011 Elsevier Inc. All rights reserved.

Assessment of Preemptive Analgesia Efficacy in Surgical Extraction of Third Molars

Background and objectives: Literature on preemptive analgesia is controversial. Reliability of results and difficult reproducibility of research contribute for non-elucidation of the subject. The aim of this study is to test the efficacy of oral ketoprofen (150 mg) preemptively administrated two days before third molar surgery, compared with postoperative administration in the same patient. Methods: Thirteen patients underwent surgical removal of bilateral third molar in two separate procedures. In a random and double blind procedure, oral ketoprofen 150 mg was administered every 12 hours two days before surgery and, after the procedure, the same drug was administered for three days. On the other side, a control (placebo) was used orally every 12 hours two days before surgery and, after the procedure, ketoprofen 150 mg was administered every 12 hours for three days. Postoperative pain was assessed by visual analogue scale, nominal scale, and amount of rescue analgesics consumed. Results: There was no statistically significant difference in postoperative pain between the preemptive treatment and control. Conclusion: In this experimental model, preemptive analgesia was not effective in reducing postoperative pain in surgical extraction of third molar compared with the postoperative administration of the same drug.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Alternative management of diabetic ketoacidosis in a Brazilian pediatric emergency department

DKA is a severe metabolic derangement characterized by dehydration, loss of electrolytes, hyperglycemia, hyperketonemia, acidosis and progressive loss of consciousness that results from severe insulin deficiency combined with the effects of increased levels of counterregulatory hormones (catecholamines, glucagon, cortisol, growth hormone). The biochemical criteria for diagnosis are: blood glucose > 200 mg/dl, venous pH <7.3 or bicarbonate <15 mEq/L, ketonemia >3 mmol/L and presence of ketonuria. A patient with DKA must be managed in an emergency ward by an experienced staff or in an intensive care unit (ICU), in order to provide an intensive monitoring of the vital and neurological signs, and of the patient's clinical and biochemical response to treatment. DKA treatment guidelines include: restoration of circulating volume and electrolyte replacement; correction of insulin deficiency aiming at the resolution of metabolic acidosis and ketosis; reduction of risk of cerebral edema; avoidance of other complications of therapy (hypoglycemia, hypokalemia, hyperkalemia, hyperchloremic acidosis); identification and treatment of precipitating events. In Brazil, there are few pediatric ICU beds in public hospitals, so an alternative protocol was designed to abbreviate the time on intravenous infusion lines in order to facilitate DKA management in general emergency wards. The main differences between this protocol and the international guidelines are: intravenous fluid will be stopped when oral fluids are well tolerated and total deficit will be replaced orally; if potassium analysis still indicate need for replacement, it will be given orally; subcutaneous rapid-acting insulin analog is administered at 0.15 U/kg dose every 2-3 hours until resolution of metabolic acidosis; approximately 12 hours after treatment initiation, intermediate-acting (NPH) insulin is initiated at the dose of 0.6-1 U/kg/day, and it will be lowered to 0.4-0.7 U/kg/day at discharge from hospital.

The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin

Objectives. To evaluate if the incorporation of antimicrobial compounds to chelating agents or the use of chelating agents with antimicrobial activity as 7% maleic acid and peracetic acid show similar disinfection ability in comparison to conventional irrigants as sodium hypochlorite or iodine potassium iodide against biofilms developed on dentin. Materials and methods. The total bio-volume of live cells, the ratio of live cells and the substratum coverage of dentin infected intra-orally and treated with the irrigant solutions: MTAD, Qmix, Smear Clear, 7% maleic acid, 2% iodine potassium iodide, 4% peracetic acid, 2.5% and 5.25% sodium hypochlorite was measured by using confocal microscopy and the live/dead technique. Five samples were used for each irrigant solution. Results. Several endodontic irrigants containing antimicrobials as clorhexidine (Qmix), cetrimide (Smear Clear), maleic acid, iodine compounds or antibiotics (MTAD) lacked an effective antibiofilm activity when the dentin was infected intra-orally. The irrigant solutions 4% peracetic acid and 2.5–5.25% sodium hypochlorite decrease significantly the number of live bacteria in biofilms, providing also cleaner dentin surfaces (p < 0.05). Conclusions. Several chelating agents containing antimicrobials could not remove nor kill significantly biofilms developed on intra-orally infected dentin, with the exception of sodium hypochlorite and 4% peracetic acid. Dissolution ability is mandatory for an appropriate eradication of biofilms attached to dentin.