Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Highly efficient ketone body treatment in multiple acyl-CoA dehydrogenase deficiency-related leukodystrophy.

BACKGROUND Multiple acyl-CoA dehydrogenase deficiency- (MADD-), also called glutaric aciduria type 2, associated leukodystrophy may be severe and progressive despite conventional treatment with protein- and fat-restricted diet, carnitine, riboflavin, and coenzyme Q10. Administration of ketone bodies was described as a promising adjunct, but has only been documented once. METHODS We describe a Portuguese boy of consanguineous parents who developed progressive muscle weakness at 2.5 y of age, followed by severe metabolic decompensation with hypoglycaemia and coma triggered by a viral infection. Magnetic resonance (MR) imaging showed diffuse leukodystrophy. MADD was diagnosed by biochemical and molecular analyses. Clinical deterioration continued despite conventional treatment. Enteral sodium D,L-3-hydroxybutyrate (NaHB) was progressively introduced and maintained at 600 mg/kg BW/d (≈3% caloric need). Follow up was 3 y and included regular clinical examinations, biochemical studies, and imaging. RESULTS During follow up, the initial GMFC-MLD (motor function classification system, 0 = normal, 6 = maximum impairment) level of 5-6 gradually improved to 1 after 5 mo. Social functioning and quality of life recovered remarkably. We found considerable improvement of MR imaging and spectroscopy during follow up, with a certain lag behind clinical recovery. There was some persistent residual developmental delay. CONCLUSION NaHB is a highly effective and safe treatment that needs further controlled studies.

Omega-3 poly-unsaturated fatty acids for the prevention of severe neutropenic enterocolitis in patients with acute myeloid leukemia

Neutropenic enterocolitis is a potentially fatal complication of myeloablative chemotherapy in patients with acute myeloid leukemia. Omega-3 polyunsaturated fatty acids (PUFA) are precursors of potent anti-inflammatory prostaglandins. Our aim was to explore the safety and effectiveness of omega-3 PUFA added to parenteral nutrition in protecting leukemia patients from severe enterocolitis. Fourteen patients with acute myeloid leukemia who received omega-3 PUFA in a Phase II trial were compared with 66 consecutive control patients not getting this intervention. We performed crude and adjusted comparisons, using inverse probability of treatment weighting for adjusted analysis, and blind outcome assessment to minimize assessor bias. Primary outcome was severe enterocolitis (≥Grade 3). The crude odds ratio of Grade 3 colitis or higher was 1.36 (95% CI 0.37 to 4.96, P = 0.64), and the adjusted odds ratio was 0.79 (95% CI 0.35 to 1.78, P = 0.57). There was little evidence to suggest differences between groups in serious adverse events and overall mortality. Our results provide little evidence that addition of omega-3 PUFA is beneficial in this condition. Routine treatment with omega-3 PUFA is currently not warranted.

Toll-like receptor-3-induced mitochondrial dysfunction in cultured human hepatocytes

Several studies have shown the presence of liver mitochondrial dysfunction during sepsis. TLR3 recognizes viral double-stranded RNA and host endogenous cellular mRNA released from damaged cells. TLR3 ligand amplifies the systemic hyperinflammatory response observed during sepsis and in sepsis RNA escaping from damaged tissues/cells may serve as an endogenous ligand for TLR3 thereby modulating immune responses. This study addressed the hypothesis that TLR3 might regulate mitochondrial function in cultured human hepatocytes. HepG2 cells were exposed to TLR-3 ligand (dsRNA--polyinosine-polycytidylic acid; Poly I:C) and mitochondrial respiration was measured. Poly I:C induced a reduction in maximal mitochondrial respiration of human hepatocytes which was prevented partially by preincubation with cyclosporine A (a mitochondrial permeability transition pore-opening inhibitor). Poly-I:C induced activation of NF-κB, and the mitochondrial dysfunction was accompanied by caspase-8 but not caspase-3 activation and by no major alterations in cellular or mitochondrial ultrastructure.

Pabp binds to the osk 3'UTR and specifically contributes to osk mRNA stability and oocyte accumulation

RNA localization is tightly coordinated with RNA stability and translation control. Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors are part of a Drosophila transport machinery that localizes mRNAs to specific cellular regions during oogenesis and embryogenesis. We identified the Poly(A)-binding protein (Pabp) as a protein that forms an RNA-dependent complex with Bic-D in embryos and ovaries. pabp also interacts genetically with Bic-D and, similar to Bic-D, pabp is essential in the germline for oocyte growth and accumulation of osk mRNA in the oocyte. In the absence of pabp, reduced stability of osk mRNA and possibly also defects in osk mRNA transport prevent normal oocyte localization of osk mRNA. pabp also interacts genetically with osk and lack of one copy of pabp(+) causes osk to become haploinsufficient. Moreover, pointing to a poly(A)-independent role, Pabp binds to A-rich sequences (ARS) in the osk 3'UTR and these turned out to be required in vivo for osk function during early oogenesis. This effect of pabp on osk mRNA is specific for this RNA and other tested mRNAs localizing to the oocyte are less and more indirectly affected by the lack of pabp

Mechanical and radiological assessment of the influence of rhTGFbeta-3 on bone regeneration in a segmental defect in the ovine tibia: pilot study

Limitations in the use of autologous bone graft, which is the gold standard therapy in bone defect healing, drive the search for alternative treatments. In this study the influence of rhTGFbeta-3 on mechanical and radiological parameters of a healing bone defect in the sheep tibia was assessed. In the sheep, an 18-mm long osteoperiosteal defect in the tibia was treated by rhTGFbeta-3 seeded on a poly(L/DL-lactide) carrier (n = 4). In a second group (n = 4), the defect was treated by the carrier only, in a third group (n = 4) by autologous cancellous bone graft, and in a fourth group (n = 2) the defect remained blank. The healing process of the defect was assessed by weekly in vivo stiffness measurements and radiology as well as by quantitative computed tomographic assessment of bone mineral density (BMD) every 4 weeks. The duration of the experiment was 12 weeks under loading conditions. In the bone graft group, a marginally significant higher increase in stiffness was observed than in the PLA/rhTGFbeta-3 group (p = 0.06) and a significantly higher increase than in the PLA-only group (p = 0.03). The radiographic as well as the computed tomographic evaluation yielded significant differences between the groups (p = 0.03), indicating the bone graft treatment (bone/per area, 83%; BMD, 0.57 g/cm(3)) performing better than the PLA/rhTGFbeta-3 (38%; 0.23 g/cm(3)) and the PLA-only treatment (2.5%; 0.09 g/cm(3)), respectively. Regarding the mechanical and radiological parameters assessed in this study, we conclude that rhTGFbeta-3 has a promoting effect on bone regeneration. However, under the conditions of this study, this effect does not reach the potential of autologous cancellous bone graft transplantation.

Pharmacokinetics and excretion of gamma-hydroxybutyrate (GHB) in healthy subjects

In Europe and the United States, the recreational use of gamma-hydroxy butyric acid (GHB) at dance clubs and "rave" parties has increased substantially. In addition, GHB is used to assist in the commission of sexual assaults. The aim of this controlled clinical study was to acquire pharmacokinetic profiles, detection times, and excretion rates in human subjects. Eight GHB-naïve volunteers were administered a single 25-mg/kg body weight oral dose of GHB, and plasma, urine, and oral fluid specimens were analyzed by using gas chromatography-mass spectrometry (GC-MS). Liquid-liquid extraction was performed after acid conversion of GHB to gamma-butyrolactone. Limits of quantitation of 0.1 (oral fluid), 0.2 (urine), and 0.5 microg/mL (plasma) could be achieved in the selected ion monitoring mode. GHB plasma peaks of 39.4 +/- 25.2 microg/mL (mean +/- SEM) occurred 20-45 min after administration. The terminal plasma elimination half-life was 30.4 +/- 2.45 min, the distribution volume 52.7 +/- 15.0 L, and the total clearance 1228 +/- 233 microL/min. In oral fluid, GHB could be detected up to 360 min, with peak concentrations of 203 +/- 92.4 microg/mL in the 10-min samples. In urine, 200 +/- 71.8 and 230 +/- 86.3 microg/mL, were the highest GHB levels measured at 30 and 60 min, respectively. Only 1.2 +/- 0.2% of the dose was excreted, resulting in a detection window of 720 min. Common side-effects were confusion, sleepiness, and dizziness; euphoria and change of vital functions were not observed. GHB is extensively metabolized and rapidly eliminated in urine and oral fluid. Consequently, samples should be collected as soon as possible after ingestion.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Long-term elevation of β-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma β-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-β-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Osteo-odonto-keratoprosthesis (OOKP) and the testing of three different adhesives for bonding bovine teeth with optical poly-(methyl methacrylate) (PMMA) cylinder.

AIM Preparation of the lamina during osteo-odonto-keratoprosthesis (OOKP) design is complex, and its longevity and watertightness important. To date, only acrylic bone cements have been used for bonding the optical cylinder to the tooth dentine. Our aim was to evaluate different dental adhesives for OOKP preparation. METHODS Specimens of bovine teeth were produced by preparing 1.5-mm thick dentine slices with holes having a diameter of 3.5 mm. Each group (n=10 per group) was luted with either classic poly-(methyl methacrylate) (PMMA) bone cement, universal resin cement or glass ionomer cement. All specimens underwent force measurement using a uniaxial traction machine. RESULTS The highest mean force required to break the bond was measured for PMMA bone cement (128.2 N) followed by universal resin cement (127.9 N), with no statistically significant difference. Glass ionomer cement showed significantly lower force resistance (78.1 N). CONCLUSIONS Excellent bonding strength combined with easy application was found for universal resin cement, and thus, it is a potential alternative to acrylic bone cement in OOKP preparation.