Farnesoid X receptor agonism - a new approach to the treatment of cholesterol gallstone disease

Gallstone disease is very common among native Americans and Hispanics, and similar to 20 million patients are treated for this disease annually in the US. The nuclear farnesoid X receptor (FXR) is the receptor for bile acids, and GW4064 is a synthetic agonist at the FXR. FXR-/- mice fed a lithogenic diet (high fat, cholesterol and cholic acid) are more susceptible to gallstone disease than wild-type mice with the same mixed background, thus establishing that the ablation of FXR is associated with this disease. The C57L mouse is susceptible to gallstone formation. When C57L mice are fed a lithogenic diet for a week, the bile contains large aggregates of cholesterol precipitates, and two of five C57L mice had macroscopic cholesterol crystals. in contrast, when C57L mice were fed the lithogenic diet and administered GW4064 100 mg/kg/day by oral gavage, there was no precipitation of cholesterol. Treatment with this agent also increased bile salt and phospholipid concentration, and prevented gallbladder epithelium damage. As FXR agonism with GW4064 has been shown to be useful in a mouse model of cholesterol gallstone disease, it should undergo further development for the treatment of this condition.

Formulation design considerations for oral vaccines

Whilst oral vaccination is a potentially preferred route in terms of patient adherence and mass vaccination, the ability to formulate effective oral vaccines remains a challenge. The primary barrier to oral vaccination is effective delivery of the vaccine through the GI tract owing to the many obstacles it presents, including low pH, enzyme degradation and bile-salt solubilization, which can result in breakdown/deactivation of a vaccine. For effective immune responses after oral administration, particulates need to be taken up bythe M cells however, these are few in number. To enhance M-cell uptake, particle characteristics can be optimized with particle size, surface charge, targeting groups and bioadhesive properties all being considerations. Yet improved uptake may not translate into enhanced immune responses and formulating particulates with inherent adjuvant properties can offer advantages. Within this article, we establish the options available for consideration when building effective oral particulate vaccines.

Histidine acts as a co-germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co-germinant. The aim of this study was to determine whether any other amino acids act as co-germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re-germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co-germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l-1. Conclusions: Histidine was found to be a co-germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Bile And Liver Metallothionein Behavior In Copper-exposed Fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Effect Of Composite Containing An Iodonium Salt On The Bond Strength Of Brackets To Bovine Enamel.

This study investigated the effect of the incorporation of an iodonium salt in experimental composites, on the bond strength of metallic brackets bonded to bovine teeth. Two hundred and seventy bovine teeth were embedded in self-curing acrylic resin and divided into 18 groups (n=15), according to the experimental composite with an iodonium salt at molar concentrations 0 (control), 0.5, or 1%; the light-activation times (8, 20 and 40 s); and the storage times (10 min or 24 h). Metallic brackets were fixed on the tooth surface using experimental composites. Photoactivation was performed with a quartz-tungsten-halogen light-curing unit curing unit for 8, 20 and 40 s. The specimens were stored in distilled water at 37 °C for 10 min or 24 h and submitted to bond strength test at 0.5 mm/min. The data were subjected to three-way ANOVA and Tukey's test (α=0.05). The Adhesive Remnant Index (ARI) was used to classify the failure modes. The shear bond strengths (MPa) at 10 min for light-activation times of 8, 20 and 40 s were: G1 - 4.6, 6.9 and 7.1; G2 - 8.1, 9.2 and 9.9; G3 - 9.1, 10.4 and 10.7; and at 24 h were: G1 - 10.9, 11.1 and 11.7; G2 - 11.8, 12.7 and 14.2; G3 - 12.1, 14.4 and 15.8. There was a predominance of ARI score 3 for groups with 10 min storage time, and ARI score 2 for groups with 24 h storage time. In conclusion, the addition of iodonium salt (C05 and C1) to the experimental composite may increase the bond strength of brackets to bovine enamel using reduced light exposure times.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

On the stabilization of conducting pernigraniline salt by the synthesis and oxidation of polyaniline in hydrophobic ionic liquids

The electrochemical polymerization of aniline in a hydrophobic room-temperature ionic liquid and the spectroelectrochemical characterization of the formed film are presented. The polymerization occurs without the presence of acid in 1-butyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide (BMMITFSI), leading to a very stable electroactive material where no degradation was observed even at high applied potentials. Both in situ UV-Vis and Raman spectroscopic studies provided evidence for the stabilization of pernigraniline salt at high oxidation potentials and that this polyaniline state is the conducting form, as was corroborated by in situ resistance measurements. These data are indicative that low conductivity is not an intrinsic property of pernigraniline salt and this point must be reconsidered.

Downscaling a multicommuted flow injection analysis system for the photometric determination of iodate in table salt

In this work a downscaled multicommuted flow injection analysis setup for photometric determination is described. The setup consists of a flow system module and a LED based photometer, with a total internal volume of about 170 mu L The system was tested by developing an analytical procedure for the photometric determination of iodate in table salt using N,N-diethyl-henylenediamine (DPD) as the chromogenic reagent. Accuracy was accessed by applying the paired r-test between results obtained using the proposed procedure and a reference method, and no significant difference at the 95% confidence level was observed. Other profitable features, such as a low reagent consumption of 7.3 mu g DPD per determination: a linear response ranging from 0.1 up to 3.0 m IO(3)(-), a relative standard deviation of 0.9% (n = 11) for samples containing 0.5 m IO(3)(-), a detection limit of 17 mu g L(-1) IO(3)(-), a sampling throughput of 117 determination per hour, and a waste generation 600 mu L per determination, were also achieved. (C) 2010 Elsevier B.V. All rights reserved.

Morphological analysis of pitting corrosion in AISI 310S austenitic stainless steel after salt spray exposition

This paper aims to study evolution of increase, distribution and classification of pits in 310S austenitic stainless steels obtained in the state as-received and heat-treated under different exposure times in saline. This work applicability has been based on a technique development for morphologic characterization of localized corrosion associated with description aspects of shapes, size and population-specific parameters. Methodology has been consisted in the following steps: specimens preparation, corrosion tests via salt spray in different conditions, microstructural analysis, pits profiles analysis and images analysis, digital processing and image analysis in order to characterize the pits distribution, morphology and size. Results obtained in digital processing and profiles image analysis have been subjected to statistical analysis using median as parameter in the alloy as received and treated. The alloy as received displays the following morphology: hemispheric pits> transition region A> transition region B> irregular> conic. The pits amount in the treated alloy at each exposure time is: transition region B> hemispherical> transition region A> conic> irregular.

Influence of the Type of Quaternary Ammonium Salt Used in the Organic Treatment of Montmorillonite on the Properties of Poly(Styrene-co-Butyl Acrylate)/Layered Silicate Nanocomposites Prepared by In Situ Miniemulsion Polymerization

Hybrid latices of poly(styrene-co-butyl acrylate) were synthesized via in situ miniemulsion polymerization in the presence of 3 and 6 wt % organically modified montmorillonite (OMMT). Three different ammonium salts: cetyl trimethyl ammonium chloride (CTAC), alkyl dimethyl benzyl ammonium chloride (Dodigen), and distearyl dimethyl ammonium chloride (Praepagen), were investigated as organic modifiers. Increased affinity for organic liquids was observed after organic modification of the MMT. Stable hybrid latices were obtained even though miniemulsion stability was disturbed to some extent by the presence of the OMMTs during the synthesis. Highly intercalated and exfoliated polymer-MMT nanocomposites films were produced with good MMT dispersion throughout the polymeric matrix. Materials containing MMT modified with the 16 carbons alkyl chain salt (CTAC) resulted in the largest increments of storage modulus, indicating that single chain quaternary salts provide higher increments on mechanical properties. Films presenting exfoliated structure resulted in the largest increments in the onset temperature of decomposition. For the range of OMMT loading studied, the nanocomposite structure influenced more significantly the thermal stability properties of the hybrid material than did the OMMT loading. The film containing 3 wt % MMT modified with the two 18 carbons alkyl chains salt (Praepagen) provided the highest increment of onset temperature of decomposition. (C) 2010 Wiley Periodicals, Inc. J Appl Polym Sci 119: 3658-3669, 2011

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4-beta-N-acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg(-1), whereas cellulase complex (1,4-(1,3 : 14)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for alpha-amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. (C) 2010 Society of Chemical Industry

Phase equilibria of lysozyme precipitation with the volatile salt ammonium carbamate

Lysozyme precipitation induced by the addition of the volatile salt ammonium carbamate was studied through cloud-point measurements and precipitation assays. Phase equilibrium experiments were carried out at 5.0, 15.0 and 25.0 degrees C and the compositions of the coexisting phases were determined. A complete separation of the coexisting liquid and solid phases could not be achieved. Nevertheless it was possible to determine the composition of the solid precipitate through the extensions of experimental tie lines. The same precipitate was found at all temperatures. Lysozyme enzymatic activities of the supernatant and precipitate phases were also determined. The activity balance suggests that ammonium carbamate preserves lysozyme activity after the salting-out precipitation. (C) 2010 Elsevier B.V. All rights reserved.

Phase equilibria for salt-induced lysozyme precipitation: Effect of salt type and temperature

The salt-induced precipitation of lysozyme from aqueous solutions was studied through precipitation assays in which the equilibrium compositions of the coexisting phases were determined. Lysozyme precipitation experiments were carried out at 5, 15 and 25 degrees C and pH 7.0 with ammonium sulfate, sodium sulfate and sodium chloride as precipitating agents. In these experiments a complete separation of the coexisting phases (liquid and solid) could not be achieved. Nevertheless it was possible to determine the composition of the precipitate. The enzymatic activity of lysozyme in the supernatant phase as well as in the precipitate phase was also determined. The activity balance suggests that there is a relationship between the composition of the true precipitate and the total activity recovery. (C) 2009 Elsevier B.V. All rights reserved.