A forced titration study of the antioxidant and immunomodulatory effects of Ambrotose AO supplement

Background Oxidative stress plays a role in acute and chronic inflammatory disease and antioxidant supplementation has demonstrated beneficial effects in the treatment of these conditions. This study was designed to determine the optimal dose of an antioxidant supplement in healthy volunteers to inform a Phase 3 clinical trial. Methods The study was designed as a combined Phase 1 and 2 open label, forced titration dose response study in healthy volunteers (n = 21) to determine both acute safety and efficacy. Participants received a dietary supplement in a forced titration over five weeks commencing with a no treatment baseline through 1, 2, 4 and 8 capsules. The primary outcome measurement was ex vivo changes in serum oxygen radical absorbance capacity (ORAC). The secondary outcome measures were undertaken as an exploratory investigation of immune function. Results A significant increase in antioxidant activity (serum ORAC) was observed between baseline (no capsules) and the highest dose of 8 capsules per day (p = 0.040) representing a change of 36.6%. A quadratic function for dose levels was fitted in order to estimate a dose response curve for estimating the optimal dose. The quadratic component of the curve was significant (p = 0.047), with predicted serum ORAC scores increasing from the zero dose to a maximum at a predicted dose of 4.7 capsules per day and decreasing for higher doses. Among the secondary outcome measures, a significant dose effect was observed on phagocytosis of granulocytes, and a significant increase was also observed on Cox 2 expression. Conclusion This study suggests that Ambrotose AO® capsules appear to be safe and most effective at a dosage of 4 capsules/day. It is important that this study is not over interpreted; it aimed to find an optimal dose to assess the dietary supplement using a more rigorous clinical trial design. The study achieved this aim and demonstrated that the dietary supplement has the potential to increase antioxidant activity. The most significant limitation of this study was that it was open label Phase 1/Phase 2 trial and is subject to potential bias that is reduced with the use of randomization and blinding. To confirm the benefits of this dietary supplement these effects now need to be demonstrated in a Phase 3 randomised controlled trial (RCT)

Rapidity and Centrality Dependence of Proton and Anti-proton Production from Au+Au Collisions at sqrt(sNN) = 130GeV

We report on the rapidity and centrality dependence of proton and anti-proton transverse mass distributions from Au+Au collisions at sqrt(sNN) = 130GeV as measured by the STAR experiment at RHIC. Our results are from the rapidity and transverse momentum range of |y|<0.5 and 0.35 <p_t<1.00GeV/c. For both protons and anti-protons, transverse mass distributions become more convex from peripheral to central collisions demonstrating characteristics of collective expansion. The measured rapidity distributions and the mean transverse momenta versus rapidity are flat within |y|<0.5. Comparisons of our data with results from model calculations indicate that in order to obtain a consistent picture of the proton(anti-proton) yields and transverse mass distributions the possibility of pre-hadronic collective expansion may have to be taken into account.Comment: 4 pages, 3 figures, 1 table, submitted to PR

A Systematic Analysis of Eluted Fraction of Plasma Post Immunoaffinity Depletion: Implications in Biomarker Discovery

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at <1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample

A new technique for seeding chondrocytes onto solvent-preserved human meniscus using the chemokinetic effect of recombinant human bone morphogenetic protein-2

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients’ cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins

Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies

Gene expression profile of peripheral blood lymphocytes from renal cell carcinoma patients treated with IL-2, Interferon-α and dendritic cell vaccine

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e50221, doi:10.1371/journal.pone.0050221.Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and TREG-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of TREG-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.This work was supported by grants from the National Institutes of Health (RO1 CA5648, R21CA112761, P20RR016437, and P30CA023108)

Chromium Stress Mitigation by Polyamine-Brassinosteroid Application Involves Phytohormonal and Physiological Strategies in Raphanus sativus L.

Brassinosteroids (BRs) and polyamines (PAs) are well-established growth regulators playing key roles in stress management among plants. In the present study, we evaluated the effects of epibrassinolide (EBL, an active BR) and spermidine (Spd, an active PA) on the tolerance of radish to oxidative stress induced by Cr (VI) metal. Our investigation aimed to study the impacts of EBL (10−9 M) and/or Spd (1 mM) on the biochemical and physiological responses of radish (Raphanus sativus L.) under Cr-stress. Applications of EBL and/or Spd were found to improve growth of Cr-stressed seedlings in terms of root length, shoot length and fresh weight. Our data also indicated that applications of EBL and Spd have significant impacts, particularly when applied together, on the endogenous titers of PAs, free and bound forms of IAA and ABA in seedlings treated with Cr-stress. Additionally, co-applications of EBL and Spd modulated more remarkably the titers of antioxidants (glutathione, ascorbic acid, proline, glycine betaine and total phenol) and activities of antioxidant enzymes (guaicol peroxidase, catalase, superoxide dismutase and glutathione reductase) in Cr-stressed plants than their individual applications. Attenuation of Cr-stress by EBL and/or Spd (more efficient with EBL and Spd combination) was also supported by enhanced values of stress indices, such as phytochelatins, photosynthetic pigments and total soluble sugars, and reduction in malondialdehyde and H2O2 levels in Cr-treated seedlings. Diminution of ROS production and enhanced ROS scavenging capacities were also noted for EBL and/or Spd under Cr-stress. However, no significant reduction in Cr uptake was observed for co-application of EBL and Spd when compared to their individual treatments in Cr-stressed seedlings. Taken together, our results demonstrate that co-applications of EBL and Spd are more effective than their independent treatments in lowering the Cr-induced oxidative stress in radish, leading to improved growth of radish seedlings under Cr-stress