Inter-day accuracy and precision were also assessed from the analysis of the same QC samples on three separate occasions in replicate (n = 6). QC samples were analyzed against calibration standards. During the course of study the probability of encountering samples with concentrations above the upper limit of quantitation (ULOQ) could not be ruled out and therefore dilution with drug free plasma is necessary to bring them within the calibration range. To establish the effect of dilution on the integrity of samples, six aliquots buy DAPT of 63001.36 ng/mL and 12370.35 ng/mL of AMX and CLV respectively were prepared.

The samples were subjected to twofold dilution (n = 6) and fivefold dilution (n = 6) with drug free human plasma to bring them within the Kinase Inhibitor Library clinical trial calibration range. The samples were processed, analyzed and the concentrations obtained were compared with theoretical values. Evaluation of the stability of samples was based on the comparison of various samples against freshly prepared samples of the same concentration. Percentage difference between the back calculated concentrations obtained for the sample under investigation

and freshly prepared sample was evaluated. Six aliquots, each of LQC and HQC concentrations were used for stability study. Bench top stability was studied on samples kept at ambient temperature (20–30 °C) for 6 h 26 min. The processed samples were kept in the autosampler at 5 °C for 59 h 33 min and then injected to determine the stability in the autosampler. The freeze–thaw stability of samples stored at −80 °C was studied after subjecting the samples to five freeze–thaw cycles. The long term stability of the samples were determined after storing the samples at −80 °C for 28 days. In order to determine the stability of AMX and CLV in solution, the working solution was kept at 2–8 °C for 9 days 22 h. Thereafter, Endonuclease the mean areas of AMX and CLV from six

replicate chromatographic runs were compared to that of the mean area of a freshly prepared solution of the same concentration. For the pharmacokinetic studies co-amoxiclav a single dose of 875/125 mg tablets was administered orally. 24 healthy adult male volunteers who gave written informed consent took part in this study. The study was approved by Ethics Committee of Institutional Review Board. The volunteers were selected on predetermined inclusion/exclusion criteria. Males had a mean age of 27.19 ± 6.32 years, mean weight of 60.87 ± 7.07 kg, mean height of 167.87 ± 5.53 cm and a body mass index mean of 21.57 ± 1.93 kg/cm2. The volunteers who were included in the study have not taken any other medication for at least two weeks beforehand. Blood samples were taken by using vacutainers, precoated with sodium heparin, at 0.00, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 5.00, 6.00, 7.00, 8.00, 10.00, and 12.00 h after ingestion.

The four fractions obtained were analysed with standard screening tests to detect the principal secondary metabolites. From residues of the ethanol extractions lipids were extracted with chloroform–methanol (2:1).12 Flavonoids were analysed using planar chromatography with two different mobile phases

(BAW: n-butanol–acetic acid–water, 4:1:5; Forestal: acetic acid–conc. HCl–water, 30:3:10). For lipids, a one-dimensional system was used on Silica gel G60 impregnated with ammonium sulphate, with benzene–acetone–water (30:91:8) as mobile phase.13 Pigments were determined from the soluble fractions in dichloromethane in Silica gel G60-calcium carbonate (2:1) with petroleum ether–acetone–i-propanol (35.5:14:0.5) used as mobile phase. 14 Furthermore, the second exhaustive extraction Selleck Dabrafenib of pigments was performed using acetone and MgCO3 to avoid the accidental formation of chlorophyll metabolites. The extracts were centrifuged at 670 × g, dried under vacuum and resuspended in 500 μl of acetone. The extracts where analysed by HPLC-RP-DAD. 15 The pigments were identified by co-chromatography with appropriate standards during elution, and by comparing their absorption spectra with reference standards. Standards and extracts were run through a C18 column, using a solution of acetonitrile: water (90:10) as mobile phase, at 1 ml/min flow rate and readings

were taken at 436 nm. selleckchem The scavenging activity on diphenyl-2-picryl hydrazyl (DPPH) radicals of ethanolic and dichloromethane fractions (A and B respectively, Fig. 1) was assayed. The radical scavenging activities expressed next as percentage inhibition of DPPH were calculated.16 The SC50 values

were calculated by linear regression.17 Only high polar extracts (Fraction A) were analysed by the wheat rootlet growth inhibition bioassay (Triticum sativum) 18 since assay requires the sample to be soluble in water. Vinblastine sulphate was used as a positive control. The toxicity of the extracts was monitored by the brine shrimp lethality test.19 The efficiency of biomass production and the four fractions obtained is shown in Tables 1 and 2. The phytochemical screening showed in all samples the presence of carbohydrates of low molecular weight, lipids, and steroids. Cardenolids were only present in the exponential phase samples, and triterpenes only in the exponential phase samples of the bleached strains. With the exception of the MAT (ph), tannins were present in the exponential phase of all the other samples. In contrast, flavonoids were only detected in the stationary phase samples of photosynthetic strains (Table 3). The presence in all photosynthetic samples of chlorophylls a, b; β, β-carotenes; diadinoxanthin and neoxanthin was verified by TLC. The second analysis performed by RP-HPLC-DAD allowed yields between 33% (UTEX-h-ST) and 68.8% (MAT-ph-ST). Table 4 shows for each pigment detected the retention times (RT), the real absorption maxima in the elution solvent, and the extraction yields.

In the absence of transporter inhibition, ambient [Glu] has been reported as being too low to activate AMPA receptors,

selleck chemicals even when desensitization is pharmacologically blocked (Le Meur et al., 2007). In contrast, ambient [Glu] has been reported to tonically activate high-affinity NMDA receptors (Sah et al., 1989, Cavelier and Attwell, 2005, Le Meur et al., 2007 and Herman and Jahr, 2007). Several patch clamp studies in acute hippocampal slice have provided estimates of ambient [Glu] based on analyses of the tonic NMDA receptor currents in CA1 pyramidal neurons. These have been reported as ∼25 nM at 32° (Herman and Jahr, 2007), 27–33 nM at 25° and 77–89 nM at 35° (Cavelier and Attwell, 2005), and 83–87 nM at 25° (Le Meur et al., 2007). These estimates are not likely to be artifactually low due to loss of glutamate from the surface of the slice, because inclusion of 2 μM glutamate in the recording chamber did not alter the level of tonic receptor activity (Herman and Jahr, 2007). The major source of glutamate in these studies was of non-vesicular origin. A range of possible molecular mechanisms may underlie glutamate release, including glutamate-permeable anion channels, the cystine-glutamate exchanger xCT, and passive membrane diffusion (Kimelberg et al., 1990, Baker

et al., 2002 and Cavelier and Attwell, 2005; for review see Cavelier et al., 2005). Elevation of ambient [Glu] by inhibition IOX1 of glutamine synthetase

suggests that a major contribution of glutamate release is from glia (Cavelier and Attwell, 2005 and Le Meur et al., 2007). The data and the diffusion model presented here suggests that a thin layer of damaged tissue with disrupted glutamate transport could underlie the significant quantitative discrepancy between the ambient glutamate estimates provided by electrophysiological studies in slices and those from microdialysis studies, which generally report ambient [Glu] values in the range ⩾2 μM (reviewed by Cavelier et al., 2005 and Featherstone and Shippy, 2008). Histological analyses of tissue surrounding microdialysis many probes provide evidence for a layer of damaged tissue up to hundreds of microns surrounding the probe (Clapp-Lilly et al., 1999, Bungay et al., 2003, Amina et al., 2003 and Jaquins-Gerstl and Michael, 2009). Diffusion modeling suggests that disrupted transport in this region could lead to artifactually large concentrations in the probe volume. A critical assumption in our model is that the glutamate leak source is constant in a volume of metabolically damaged tissue where transport is impaired. The precise spatial changes in metabolic activity in a traumatized or ischemic region of tissue are unknown, but the assumption that the leak is constant is conservative. For example, glutamate release is increased by reversed glutamate transport due to impaired Na/K gradients during metabolic challenge (Rossi et al., 2000).

The estimated vaccine effectiveness for mumps for two doses compared to one was 68% (95%CI −24% to 92%), with indications of waning immunity over time. We estimated an attack rate of mumps of 5% during this outbreak. This finding was consistent with results of several other European studies in similar settings, where the reported attack rates of mumps ranged from 1% to 7% among vaccinated populations [10] and [21]. However, in the Netherlands, during an outbreak among university students, the attack rate was higher (13%) [11]. Mandatory notification and cohort

study data suggested that the incidence was higher among http://www.selleckchem.com/products/VX-770.html males. This may have an immunological explanation. In vitro studies indicated that females have a greater immune response to vaccination than males [22]. Moreover, seroprevalence studies conducted in the Netherlands and Belgium reported lower levels of mumps-induced antibodies in males [23] and [24]. The documented vaccination coverage for two-doses of mumps-containing vaccine among our study participants was 95%. Seroprevalence studies suggest that a two-dose coverage of ≥95%for mumps protects populations from outbreaks [25] and [26]. In 2012, a vaccination coverage survey selleck in the Flemish region reported 92.5% coverage for the second dose of MMR [17]. A coverage survey, conducted

in 2005, among the birth cohort that was highly affected during the 2013 outbreak (birth year: 1991) estimated a vaccination coverage of 84% for the second dose [27]. Therefore, the vaccination coverage in Flanders may have been insufficient to protect the population against outbreaks. The low proportion of participants STK38 for whom medical files were available at the university medical service may have biased our vaccination coverage. In

our study, we could not obtain a significant vaccine effectiveness estimate. We obtained a vaccine effectiveness estimate of 68% for the second dose as compared to only one dose, indicating the benefit of vaccinating twice, but also indicating that a two dose vaccination offers incomplete protection. Results of a 2012 Cochrane review indicated a two-dose vaccine effectiveness of 83–88% for lab-confirmed cases [28]. In outbreak situations, case definitions and determination of vaccination status may influence the vaccine effectiveness estimates. Differences between the wild type virus and the vaccine strain may also explain the low vaccine effectiveness estimate in our study. Low antibody avidity to wild-type virus, as the mismatch between the vaccine genotype and that of the circulating mumps virus strains may facilitate immune escape [29]. In our study, all isolates were genotyped as G5, suggesting that this was the circulating wild type virus. Reports indicated that cross-protection between the vaccine genotype A and the circulating wild strains (mainly C, D and G) is incomplete.

The aim of this study was to obtain fundamental data in animal experiments for KSHV vaccine development. To estimate immune responses against KSHV in animals, Balb/c mice were immunized Cell Cycle inhibitor intranasally or intraperitoneally with KSHV particles, and their immunoreactions were investigated. In addition, an in vitro neutralization assay was performed using green fluorescent protein-expressing recombinant KSHV and the serum, nasal wash fluid (NW), and saliva from the KSHV-immunized mice. KSHV particles were prepared from BCBL-1 cells stimulated with phorbol 12-myristate-13 acetate (PMA; Sigma, St. Louis, MO) as described previously [26]. Briefly, BCBL-1

cells were stimulated with PMA at 20 ng/mL for 72 h. The supernatant of

BCBL-1 cells was collected and filtered through a 0.8-μm-pored membrane. Filtered supernatant was ultracentrifuged at 20,000 × g for 2 h. The pellet was dissolved in one-fiftieth volume of RPMI 1640. Virus copy number was measured with a real-time PCR as described previously [27]. A green and red fluorescent protein (GFP/RFP)-expressing recombinant KSHV, rKSHV.219 (kindly provided by Dr. Jeffrey Vieira, Washington University), was collected for the neutralization assay as described previously [28]. Female 8-week-old Balb/c mice were purchased from Clea Japan (Tokyo, Japan) and were kept under specific-pathogen-free conditions. All animal experiments were performed in accordance Cabozantinib order with the Guidelines for Animal Experiments Performed at the National Institute of Infectious Diseases (NIID) and were approved by the Animal Care and Use Committee of NIID (approvals No. 108056 and 209072). Five mice for each experimental group were anesthetized with isoflurane and immunized primarily by dropping 5 μl of phosphate buffered saline (PBS) containing

106–108 copies of KSHV or 10 ng of KSHV-encoded proteins with 10 μg of poly(I:C) (Sigma) into each nostril [29]. For immunization to the peritoneal cavity, 100-μl aliquots of PBS containing the viruses Thiamine-diphosphate kinase (106–108 copies) or proteins (100 ng) with poly(I:C) were immunized to the mice’s peritoneal cavities. Additional immunizations were performed twice, 2 and 3 weeks later. Samples of blood, spleen, and NW were obtained from mice that were sacrificed under anesthesia with isoflurane 1 week after the final immunization. NW samples were taken as previously described [17]. Saliva samples were obtained using intraperitoneal administration of pilocarpine (150 μL of 1 mg/ml in PBS per mouse, P-6503, Sigma). Copy numbers of mouse IFN-γ, CD8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were determined with real-time RT-PCR using probe-primer sets described previously [30]. Total RNA was extracted from 1 × 107 spleen cells of each mouse with Isogen RNA isolation kit (Nippon Gene, Toyama, Japan). Real-time RT-PCR was performed with one-step Quantitect probe RT-PCR kit (Qiagen, Hilden, Germany).

This could be due to removal of most proteases during the two consecutive PEG6000 precipitations of FMDV antigen. We could also detect FMDV antigen after

addition of the adjuvant by oil emulsification. Such analysis is often difficult to perform by other methods due to the difficulty in extracting the antigen from the vaccine for subsequent analysis. As a result there are only few publications about stability of vaccine antigens after addition of adjuvant. Several model protein antigens BMN 673 cell line may be structurally altered and have reduced thermal stability upon absorption to aluminium hydroxide adjuvant [22] and [23]. Here we have shown that VP4 remains associated with FMDV virions after emulsification with oil adjuvant, indicating that virions do not substantially dissociate into 12S particles due to the inclusion in an oil emulsion. This is important for vaccine efficacy since 12S particles have a 100-fold MK-2206 clinical trial reduced potency as compared to 146S particles [8]. It is known that

oil-adjuvanted FMDV O1 Manisa vaccines have reduced potency upon storage for 2 or 4 months and a complete loss of potency after 7 months storage [4]. The ability to determine various aspects of FMDV antigen integrity by SELDI-TOF-MS in oil emulsions now enables studies towards the molecular mechanism underlying such instability of FMD antigen after prolonged storage of oil emulsion vaccines. This work others was supported financially by The Netherlands Ministry of Agriculture, Nature and Food Quality. We thank Jolanda Meijlis, Peter van Bavel, Marianne Krikken, Anna Oosterbaan and Corrie van der Bijl (all Lelystad Biologicals bv.) for supplying FMDV antigens and vaccines and for valuable discussions. “
“Glioblastoma multiforme (GBM) is a devastating

primary brain tumor that causes death in ∼73% of individuals within 2 years of diagnosis despite treatment with surgery, radiation, and chemotherapy [1]. This tumor presents clinically as either primary GBM or progresses from a lower grade (WHO II or III) glioma leading to secondary GBM. Both primary and secondary GBM are WHO grade IV tumors with a similar prognosis [2]. Secondary GBM often arises from WHO grade II astrocytomas that are characterized by low cellularity, low mitotic index and a diffuse pattern of infiltration into normal brain. Due to the disseminated nature of the neoplasm, surgery and adjuvant therapies are frequently inadequate and the tumor evolves into secondary GBM within 5–10 years [2]. Gemistocytic astrocytoma (GemA) is a histological variant of astrocytoma that has been defined in an arbitrary fashion by the presence of at least 20% gemistocytes within the tumor mass [3]. Neoplastic gemistocytes are characterized by their plump appearance, slightly eosinophilic cytoplasm and eccentric nuclei. The classification of GemA has been controversial.

During Visit 3 at the hospital, the accelerometer was collected and dyspnoea level and exercise capacity were measured. Qualitative analysis: Responses during the interviews were coded into categories using the inductive content analysis approach. The aim of this qualitative research technique is to attain a condensed and broad description of a phenomenon ( Elo and Kyngas 2008). The outcome of the inductive content analysis is categories describing the investigated phenomenon. The approach includes an iterative process of open coding, creating Selleck Navitoclax categories and abstraction ( Elo and Kyngas 2008). Each interview transcript was read several times, and afterwards keywords

in the text were labelled with codes and grouped into similar concepts, after which categories Selleck BVD523 were formed. To increase consensus, the coding process was performed separately by two trained investigators (JH and MG) with the results compared and discussed afterwards. Disagreements were resolved through

discussion with the other authors. The investigators did not have any information on the measured physical activity level of the participants during the qualitative analysis. Quantitative analysis: We combined the qualitative analysis with a quantitative analysis so as to assess the relationship between the perceived reasons to be sedentary or active and the measured physical activity level. In order to assess whether any relationship exists between the qualitatively obtained categories and the objectively measured physical activity level, a k-means cluster analysis was performed. Cluster analysis is a descriptive heptaminol statistical method that attempts to identify relatively homogeneous groups of people based on their characteristics. All categories obtained from the interview were entered in the cluster analysis together with the measured physical activity level (mean steps per day). The flow of participants through the study is presented in Figure 1. In total 118 people with COPD were willing to participate, provided

informed consent, and met the eligibility criteria of the study. Three participants dropped out during the study due to lack of time or health problems. Therefore 115 participants were interviewed and performed all other measurements and were included in the qualitative analysis. Two participants wore the accelerometer less than 4 days due to mechanical problems with the accelerometer and therefore 113 participants were included in the k-means cluster analysis. The participants’ characteristics are shown in Table 1. Participants were predominantly male (68%), with mild to very severe COPD, and with a mean MMRC dyspnoea score of 1.4. Participants walked a median of 5552 steps per day. Among the participants, 28% reported that they should be more physically active, 47% reported that they were sufficiently active, and 25% reported that they were not able to be more physically active due to health problems.

3). It can also be seen from Fig. 3 that the confidence intervals of the means for the D2 dilutions were always higher than those for the D1 dilutions, independent of the aliquots, showing that the variability of the mean for dilution D2 was higher than for dilution D1, which means that the errors made in dilution D2 were greater than in D1. When the variance in the data on CFU/mL was assessed using the F-test, when different aliquots with the same dilution were compared ( Fig. 4A) the calculated F values were within the F value limits for 95%

confidence, except when aliquots 1 and 2 at dilution D1 were compared (A1D1 and A2D1) from experiment 8 with no antibiotic. This means that the errors incurred during AZD2281 ic50 the dilution and colony count procedures were the same when compared between the same

dilutions. However, when different dilutions of the same aliquot were compared, the data showed different variance levels in most cases ( Fig. 4B). The calculated F values were outside the pre-established F interval at 95% confidence level. As already reported and shown in Fig. 3, the errors in the CFU/mL data were greater at dilution D2 than they were at dilution D1, with standard deviation about ten times higher in the data for dilution D2 than for dilution D1 (data not shown). This variability Z-VAD-FMK datasheet is owing to the fact that at the higher dilution (D2), between 0 and 10 colonies were counted, while at D1, between 10 and 100 colonies were counted. This being the case, only the Bay 11-7085 data on CFU/mL obtained from dilution D1 were used for calculating Φ values in the experimental design experiments. This statistical analysis shows that when the data from dilution D1

were used, the procedures for determining plasmid stability (serial dilutions and colony count) were reproducible, meaning that the CFU/mL data obtained had statistically equivalents means and variances, within a 95% confidence interval. The optimal condition as identified by the experimental design was the condition used in experiment 1 (0.1 mM IPTG and 0 μg/mL kanamycin). This condition permitted a tenfold reduction in the inducer concentration and the elimination of kanamycin from the system, keeping the protein concentration and cell growth at similar levels while also keeping plasmid stability at levels that would not harm recombinant protein production over the 4 h expression period. In order to validate the optimal condition as identified by experimental design, replications of the culture were produced under this condition (0.1 mM IPTG and 0 μg/mL kanamycin). The cultures were allowed to grow until they reached exponential growth (Abs600 nm approximately 0.7), at which point they were induced with 0.1 mM IPTG.

15 and 16 The phytochemicals selleck products induce toxicity in tumor cells either by scavenging constitutive reactive oxygen species or by generating paradoxically additional amount of free radicals resulting in the imbalance of cellular oxidative status, leading to inhibition of cell proliferation and eventually cell death.17, 18 and 19 In a recent study,20 the bark extract of S. oleosa was examined for its cytotoxic potential against different cell lines such as 502713 (colon), SW-520 (colon), HCT-13 (colon), A-549 (lungs), HEP-2 (liver), SK-NS-H (central nervous system), and IMR-32 (neuroblastoma). SRB dye assay following the method of Skehan et al 21 is used to evaluate the cytotoxic potential. The

ethyl acetate, methanol, and water extract showed a significant cytotoxicity against all SAHA HDAC manufacturer cell lines, except the IMR-32 cell line whereas hexane and chloroform extract did not show any significant inhibition against any of the cell lines. The cytotoxic potential was correlated with their hydroxyl radical scavenging potential. Hexane and chloroform extracts were found to have least hydroxyl radical scavenging ability, hence least cytotoxicity against the different cell lines. Oxygen is used for generating

metabolic energy in our body but it also produces reactive oxygen species as by product during its various reactions in the body. Reactive oxygen species are usually atoms or a group of atoms having odd (unpaired) electrons, in aerobic cells these are produced during mitochondrial electron transport and several very oxidation reactions.22

These reactive species can, react with DNA and several other biomolecules causing what is called ‘oxidative damage to DNA’ This damage causes changes in DNA such as strand breaks; changes at cross links between DNA and protein; changes at base free sites among other changes.23 Several medicinal plants, fruits, vegetables can decrease the risk of oxidative damage as they comprise of vitamins, carotenes, phenolic compounds, flavanoids, alkanoids, tannins etc. which act as chemopreventive agents.24, 25 and 26 These phytochemicals can prevent damage by their radical scavenging ability. Thind et al evaluated the hydroxyl radical scavenging potential of S. oleosa. Extracts of roots of S. oleosa with different solvents were tested for their antiproliferative activity. Methanol extract was effective against a colon cell line (SW-620), ethyl acetate against SK-NS-H (CNS cell line) and water extract against 502713 and SW-620 (colon) cell lines. Hydroxyl radical which was used to determine radical scavenging potential of extracts, was generated by Fenton’s reaction, in site-specific and non-site-specific deoxyribose degradation assays. The extracts showed radical scavenging potential following the order of inhibition at 100 μg/mL as ethyl acetate extract (67.72%) > water extract (65.68%) > methanol extract (64.32%) in site-specific assay and as methanol extract (83.38%) > ethyl acetate extract (81.

Conflicts of interest statement: There are no conflicts of interest. “
“Viral clearance of acute HBV infection depends on a rigorous CD4+ and CD8+ T-cell-mediated response directed against HBV-specific antigens that includes production of interferon (IFN)-γ [1], [2], [3] and [4]. In patients with chronic HBV infection, T-cell responses and IFN-γ production are both severely impaired, contributing to the persistence of their HBV infection [1], [3] and [4]. Currently available drugs are capable of controlling CT99021 concentration viremia but rarely eradicate the virus [5]. Therefore, to achieve a cure (defined as hepatitis B surface antigen [HBsAg] seroconversion),

new therapies targeting HBV replication and the immune system are needed [5]. GS-4774 (formerly GI-13020) is being developed to elicit an HBV-specific T-cell immune response in patients with chronic HBV infection. GS-4774 consists of heat-inactivated yeast cells that express well-conserved regions of HBV proteins, namely HBsAg, hepatitis B core antigen (HBcAg) and hepatitis B X protein (HBx) expressed as a single fusion protein. The recombinant heat-killed whole yeast platform has been previously shown to elicit a significant T-cell response upon subcutaneous administration [6]. Preclinical experiments

in mice showed that GS-4774 elicited T-cell responses specific to HBsAg, HBcAg, and HBx and stimulated HBV-specific CD8+ T-cells [7]. In cells from patients with chronic from HBV infection, GS-4774 induced IFN-γ-producing CD4+ and CD8+ T cells that, in some cases, showed marked levels of expression AZD6738 of the Lamp-1/CD107a marker of cytotoxic function [8]. These experiments suggested that GS-4774 had potential to elicit an antiviral immune response. The present work was a first-time-in-human clinical trial of GS-4774 in healthy subjects. Healthy subjects aged ≥18 years were eligible. Subjects were recruited using

a database of healthy volunteers elicited using advertisements in the community. Before enrolment, subjects had to demonstrate negative immunoglobulin (Ig) E-mediated hypersensitivity to Saccharomyces cerevisiae. Detailed exclusion criteria are provided in Supplementary File 1. All patients were negative for HBV DNA and anti-HBc antibodies. Four subjects had low-level antibodies to HBsAg below the threshold for positivity. All subjects provided informed consent prior to screening. Local Ethics Review Committees approved the study, which was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Single-site, randomized, open-label, dose-ascending, multi-arm study conducted in the USA between January and July 2013. Subjects were allocated to one of three dose groups (n = 20 per group) to receive 10, 40 or 80 yeast units (YU) (1 YU = 107 yeast cells) of study treatment.