In vivo evaluation of EPO-secreting cells immobilized in different alginate-PLL microcapsules.

Alginates are the most employed biomaterials for cell encapsulation due to their abundance, easy gelling properties and apparent biocompatibility. However, as natural polymers different impurities including endotoxins, proteins and polyphenols can be found in their composition. Several purification protocols as well as different batteries of assays to prove the biocompatibility of the alginates in vitro have been recently developed. However, little is known about how the use of alginates with different purity grade may affect the host immune response after their implantation in vivo. The present paper investigates the long-term functionality and biocompatibility of murine erythropoietin (EPO) secreting C2C12 cells entrapped in microcapsules elaborated with alginates with different properties (purity, composition and viscosity). Results showed that independently of the alginate type employed, the animals presented elevated hematocrit levels until day 130, remaining at values between 70-87%. However, histological analysis of the explanted devices showed higher overgrowth around non-biomedical grade alginate microcapsules which could be directly related with higher impurity content of this type of alginate. Although EPO delivery may be limited by the formation of a fibrotic layer around non-biomedical grade alginate microcapsules, the high EPO secretion of the encapsulated cells together with the pharmacodynamic behaviour and the angiogenic and immune-modulatory properties of EPO result in no direct correlation between the biocompatibility of the alginate and the therapeutic response obtained.     

Streptococci from root canals in teeth with apical periodontitis receiving endodontic treatment.

OBJECTIVES: The object of this study was to investigate the diversity among streptococcal species isolated from root canals in conjunction with endodontic therapy and to characterize their production of extracellular proteins. STUDY DESIGN: Consecutive root canal samples (RCS) taken as bacteriological controls during root canal treatment of teeth with apical periodontitis were analyzed in a total of 100 clinical cases. Bacteria were isolated and classified by selective media and gas liquid chromatography. Streptococcal strains were identified by carbohydrate fermentation, hydrolysis of aesculin/arginine, and production of enzymes. Releases of extracellular proteins by streptococci and Enterococcus spp in fluid culture media were examined with SDS-PAGE and 2-dimension gel electrophoresis (2 DE). Extracellular proteins produced were quantified and qualitatively analyzed. Specific proteins were targeted with Western immunoblot assays. Comparisons were made with type strains. RESULTS: Of a total of 241 bacterial strains recovered in the first samples submitted, Streptococcus gordonii, S anginosus, and S oralis were the most frequently isolated streptococci. In 49 of 89 resubmitted samples showing bacterial growth, S gordonii and S oralis still predominated among streptococci. Other common bacterial isolates were Enterococcus spp, Lactobacillus paracasei, and Olsenella uli. Quantitative and qualitative differences in extracellular protein production were observed among clinical isolates and laboratory streptococcal strains. In similar conditions for growth, S intermedius, S anginosus, S oralis, and S gordonii were strong producers of extracellular proteins (>3.0 microg/mL), while Enterococcus spp and S mutans were weak. Whole cell protein extracts showed a different profile from that of extracellular proteins. The chaperone protein DnaK was recognized to be produced extracellularly by S gordonii, S oralis, S anginosus, and S parasanguis. CONCLUSIONS: Being strong producers of extracellular proteins and by virtue of common presence in teeth undergoing endodontic therapy, S gordonii, S anginosus, and S oralis may be of pathogenic significance in posttreatment apical periodontitis.     

Highly resolved in vivo 1H NMR spectroscopy of the mouse brain at 9.4 T.

An efficient shim system and an optimized localization sequence were used to measure in vivo 1H NMR spectra from cerebral cortex, hippocampus, striatum, and cerebellum of C57BL/6 mice at 9.4 T. The combination of automatic first- and second-order shimming (FASTMAP) with strong custom-designed second-order shim coils (shim strength up to 0.04 mT/cm2) was crucial to achieve high spectral resolution (water line width of 11-14 Hz). Requirements for second-order shim strengths to compensate field inhomogeneities in the mouse brain at 9.4 T were assessed. The achieved spectral quality (resolution, S/N, water suppression, localization performance) allowed reliable quantification of 16 brain metabolites (LCModel analysis) from 5-10-microL brain volumes. Significant regional differences (up to 2-fold, P < 0.05) were found for all quantified metabolites but Asp, Glc, and Gln. In contrast, 1H NMR spectra measured from the striatum of C57BL/6, CBA, and CBA/BL6 mice revealed only small (<13%, P < 0.05) interstrain differences in Gln, Glu, Ins, Lac, NAAG, and PE. It is concluded that 1H NMR spectroscopy at 9.4 T can provide precise biochemical information from distinct regions of the mouse brain noninvasively that can be used for monitoring of disease progression and treatment as well as phenotyping in transgenic mice models.     

Contractile and endothelium-dependent dilatory responses of cerebral arteries at various extracellular magnesium concentrations

To clarify the effect of extracellular magnesium (Mg2+) on the vascular reactivity of feline isolated middle cerebral arteries, the effects of slight alterations in the Mg2+ concentration on the contractile and endothelium-dependent dilatory responses were investigated in vitro. The contractions, induced by 10(-8)-10(-5) M norepinephrine, were significantly potentiated at low Mg2+ (0.8 mM v. the normal, 1.2 mM). High (1.6 and 2.0 mM) Mg2+ exhibited an inhibitory effect on the contractile responses. No significant changes, however, in the EC50 values for norepinephrine were found. The endothelium-dependent relaxations induced by 10(-8)-10(-5) M acetylcholine were inhibited by high (1.6 and 2.0 mM) Mg2+. Lowering of the Mg2+ concentration to 0.8 mM or total withdrawal of this ion from the medium failed to alter the dilatory potency of acetylcholine. The changes in the dilatory responses also shifted the EC50 values for acetylcholine to the right. The present results show that the contractile responses of the cerebral arteries are extremely susceptible to the changes of Mg2+ concentrations. In response to contractile and endothelium-dependent dilatory agonists, Mg2+ probably affects both the calcium influx into the endothelial and smooth muscle cells as well as the binding of acetylcholine to its endothelial receptor. Since Mg2+ deficiency might facilitate the contractile but not the endothelium-dependent relaxant responses, the present study supports a role for Mg2+ deficiency in the development of the cerebral vasospasm.     

Distribution and function of cholinergic receptors in the sheep detrusor muscle

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.