Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended. © 2012 Elsevier Ltd. All rights reserved.

Reversible, fluorescence-based optical sensor for hydrogen peroxide

The preparation and characterisation are described of a robust, reversible, hydrogen peroxide optical sensor, based on the fluorescent quenching of the dye ion-pair [Ru(bpy)(3)(2+)(Ph4B-)(2)], by O-2 produced by the catalytic breakdown of H2O2, utilizing the inorganic catalyst RuO2 center dot xH(2)O. The main feature of this system is the one-pot formulation of a coating ink that, when dried, forms an active single-layer fluorescence-based H2O2 sensor, demonstrably capable of detecting H2O2 over the range of 0.01 to 1 M, with a relative standard deviation of ca. 4% and a calculated lower limit of detection of 0.1 mM. These sensors are sterilisable, using dry-heat, and stable when stored over 40 days, without exhibiting any loss in sensitivity or response characteristics.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-1) (n = 12) and the functional limit of detection as 3.2 ng g(-1) for egg (n = 6) and 11.3 ng g(-1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the ‘cold chain’, the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 oC, but not when stored at 40 oC / 75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 oC / 75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation as compared to the original formulation when stored at 40 oC /75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140

Mucosally-administered vaccine strategies are widely investigated as a promising means of preventing HIV infection. This study describes the development of liposomal gel formulations, and novel lyophilised variants, comprising HIV-1 envelope glycoprotein, CN54gp140, encapsulated within neutral, positively charged or negatively charged liposomes. The CN54gp140 liposomes were evaluated for mean vesicle diameter, polydispersity, morphology, zeta potential and antigen encapsulation efficiency before being incorporated into hydroxyethyl cellulose (HEC) aqueous gel and subsequently lyophilised to produce a rod-shaped solid dosage form for practical vaginal application. The lyophilised liposome-HEC rods were evaluated for moisture content and redispersibility in simulated vaginal fluid. Since these rods are designed to revert to gel form following intravaginal application, mucoadhesive, mechanical (compressibility and hardness) and rheological properties of the reformed gels were evaluated. The liposomes exhibited good encapsulation efficiency and the gels demonstrated suitable mucoadhesive strength. The freeze-dried liposome-HEC formulations represent a novel formulation strategy that could offer potential as stable and practical dosage form.

Density and viscosity of several pure and water-saturated ionic liquids

Densities and viscosities were measured as a function of temperature for six ionic liquids (1-butyl-3-methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-ethyl-3-methylimidazolium ethylsulfate and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide. The density and the viscosity were obtained using a vibrating tube densimeter from Anton Paar and a rheometer from Rheometrics Scientific at temperatures up to 393 K and 388 K with an accuracy of 10-3 g cm-3 and 1%, respectively. The effect of the presence of water on the measured values was also examined by studying both dried and water-saturated samples. A qualitative analysis of the evolution of density and viscosity with cation and anion chemical structures was performed. © The Royal Society of Chemistry 2006.

Synthesis of mesoporous tungsten carbide-supported platinum and its electrocatalytic activity for methanol oxidation

High activity and stability during oxidation of methanol under the relatively anode environment are two main evaluation criterias for an effective anode electrocatalyst in direct methanol fuel cell (DMFC). Mesoporous WC samples with hollow structure were prepared by gas-solid reaction at the atmosphere of CH(4)/H(2) by using airflow spray dried ammonium metatungstate (AMT). The platinum supported on this material by impregnation-vapor phase deoxidation method served as a less expensive electro anode catalyst. XRD and SEM results showed that Pt particles were well dispersed on the surface of WC. The results showed that the Pt/WC-PME exhibited an attractive catalytic activity, and methanol oxidation process in Pt/WC-PME is affected by liquid-phase mass transfer. The results also indicated that the oxidation can be improved by raising temperatures.

The prediction of intake potential and organic matter digestibility of grass silages by near infrared spectroscopy analysis of undried samples

A study was undertaken to examine a range of sample preparation and near infrared reflectance spectroscopy (NIPS) methodologies, using undried samples, for predicting organic matter digestibility (OMD g kg(-1)) and ad libitum intake (g kg(-1) W-0.75) of grass silages. A total of eight sample preparation/NIRS scanning methods were examined involving three extents of silage comminution, two liquid extracts and scanning via either external probe (1100-2200 nm) or internal cell (1100-2500 nm). The spectral data (log 1/R) for each of the eight methods were examined by three regression techniques each with a range of data transformations. The 136 silages used in the study were obtained from farms across Northern Ireland, over a two year period, and had in vivo OMD (sheep) and ad libitum intake (cattle) determined under uniform conditions. In the comparisons of the eight sample preparation/scanning methods, and the differing mathematical treatments of the spectral data, the sample population was divided into calibration (n = 91) and validation (n = 45) sets. The standard error of performance (SEP) on the validation set was used in comparisons of prediction accuracy. Across all 8 sample preparation/scanning methods, the modified partial least squares (MPLS) technique, generally minimized SEP's for both OMD and intake. The accuracy of prediction also increased with degree of comminution of the forage and with scanning by internal cell rather than external probe. The system providing the lowest SEP used the MPLS regression technique on spectra from the finely milled material scanned through the internal cell. This resulted in SEP and R-2 (variance accounted for in validation set) values of 24 (g/kg OM) and 0.88 (OMD) and 5.37 (g/kg W-0.75) and 0.77 (intake) respectively. These data indicate that with appropriate techniques NIRS scanning of undried samples of grass silage can produce predictions of intake and digestibility with accuracies similar to those achieved previously using NIRS with dried samples. (C) 1998 Elsevier Science B.V.

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.

Evaluation of in Situ DGT Measurements for Predicting the Concentration of Cd in Chinese Field-Cultivated Rice: Impact of Soil Cd:Zn Ratios

DGT (diffusive gradients in thin-films) has been proposed as a tool for predicting Cd concentrations in rice grain, but there is a lack of authenticating data. To further explore the relationship between DGT measured Cd and concentrations in rice cultivated in challenging, metal degraded, field locations with different heavy metal pollutant sources, 77 paired soil and grain samples were collected in Southern China from industrial zones, a "cancer village" impacted by mining waste and an organic farm. In situ deployments of DGT in flooded paddy rice rhizospheres were compared with a laboratory DGT assay on dried and rewetted soil. Total soil concentrations were a very poor predictor of plant uptake. Laboratory and field deployed DGT assays and porewater measurements were linearly related to grain concentrations in all but the most contaminated samples where plant toxicity occurred. The laboratory DGT assay was the best predictor of grain Cd concentrations, accommodating differences in soil Cd, pollutant source, and Cd:Zn ratios. Field DGT measurements showed that Zn availability in the flooded rice rhizospheres was greatly diminished compared to that of Cd, resulting in very high Cd:Zn ratios (0.1) compared to commonly observed values (0.005). These results demonstrate the potential of the DGT technique to predict Cd concentrations in field cultivated rice and demonstrate its robustness in a range of environments. Although, field deployments provided important details about in situ element stoichiometry, due to the inherent heterogeneity of the rice rhizosphere soils, deployment of DGT in dried and homogenized soils offers the best possibility of a soil screening tool.

Arsenic, cadmium, and lead pollution and uptake by rice (Oryza sativa L.) grown in greenhouse

Purpose: Hunan province is well-known for its extensive base-metal extraction and smelting industries. However, the legacies of excavation operations, transportation, and selective smelting activities within Hunan have resulted in the generation of large quantities of mine wastes, which will become the sources of metal contamination in the environment. Thus, there is an increasingly important health issue underlying the study of arable land pollution and transfer of As, Cd, and Pb in the paddy soil–rice system.
Materials and methods: Paddy soils collected from mining- and smelting-impacted areas in Hunan province and rice seed (Oryza sativa L. cv Jia Hua-1) were used for pot experiments under greenhouse conditions. One 30-day-old seedling was transplanted into one pot containing 5.0 kg pretreated soil. At harvest, rice grains and shoots were washed with distilled water to remove surface soil, and oven-dried at 65°C for 96 h until a constant weight was reached. Roots were washed carefully with distilled water for the next process of extracting iron plaque using dithionite–citrate–bicarbonate solution. Total concentrations of As, Cd, and Pb in soil and rice plant tissues were measured by inductively coupled plasma mass spectrometer.
Results and discussion: Total concentrations of As, Cd, and Pb in the soils collected from 12 mining- and smelting-impacted areas in Hunan province were much higher than Hunan background values and exceeded the maximum concentration limit for soils set by the Ministry of Environmental Protection. The yields of rice grain from Pb/Zn mining and smelting sites were negatively correlated to overall pollution scores. Distributions of As, Cd, and Pb in rice plant followed: root >> shoot > husk > whole grain. About 30.1–88.1% of As, 11.2–43.5% of Cd, and 14.0–33.9% of Pb were accumulated in iron plaque on root surfaces.
Conclusions: High concentrations of As, Cd, and Pb are observed in paddy soils from mining- and smelting-impacted areas in Hunan province, indicating those paddy soils suffer serious combined heavy metal contamination. In particular, Cd is the dominant contaminant followed by As and Pb in paddy soils from most locations. The distributions of As, Cd, and Pb in rice tissue were: root >> shoot > husk > whole grain. Concentrations of Pb in all whole grain and of As and Cd in 50% of whole grain samples exceeded Chinese Hygienic Standard values for food.

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typimurium Cells on a Stainless Steel Surface

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate-and low-moisture foods. © 2013, American Society for Microbiology.

Strawberries decrease atherosclerotic markers in subjects with metabolic syndrome

Strawberries have been reported to be potent antioxidants and reduce cardiovascular risk factors, such as elevated blood pressure, hyperglycemia, dyslipidemia, and inflammation in limited studies. We hypothesized that freeze-dried strawberry supplementation will improve blood pressure, impaired glucose, dyslipidemia, or circulating adhesion molecules in obese subjects with metabolic syndrome, thereby lowering cardiovascular risk factors in these subjects. Twenty-seven subjects with metabolic syndrome (2 males and 25 females; body mass index, 37.5 +/- 2.15 kg/m(2); age, 47.0 +/- 3.0 years [means +/- SE]) consumed 4 cups of freeze-dried strawberry beverage (50 g freeze-dried strawberries approximately 3 cups fresh strawberries) or equivalent amounts of fluids (controls, 4 cups of water) daily for 8 weeks in a randomized controlled trial. Anthropometrics and blood pressure measurements, assessment of dietary intakes, and fasting blood draws were conducted at screen and 8 weeks of the study. Strawberry supplementation significantly decreased total and low-density lipoprotein cholesterol (5.8 +/- 0.2 to 5.2 +/- 0.2 mmol/L and 3.5 +/- 0.2 to 3.1 +/- 0.1 mmol/L, respectively [means +/- SE], P <.05) and small low-density lipoprotein particles using nuclear magnetic resonance-determined lipoprotein subclass profile vs controls at 8 weeks (794.6 +/- 94.0 to 681.8 +/- 86.0 nmol/L [means +/- SE], P <.05). Strawberry supplementation further decreased circulating levels of vascular cell adhesion molecule-1 vs controls at 8 weeks (272.7 +/- 17.4 to 223.0 +/- 14.0 ng/mL [means +/- SE], P <.05). Serum glucose, triglycerides, high-density lipoprotein cholesterol, blood pressure, and waist circumference were not affected. Thus, short-term freeze-dried strawberry supplementation improved selected atherosclerotic risk factors, including dyslipidemia and circulating adhesion molecules in subjects with metabolic syndrome, and these results need confirmation in future trials.