Prediction of antigen-responding VHH antibodies by tracking the evolution of antibody along the time course of immunization.

Antibody maturation is the central function of the adaptive immune response. This process is driven by the repetitive selection of mutations that increase the affinity toward antigens. We hypothesized that a precise observation of this process by high-throughput sequencing along the time course of immunization will enable us to predict the antibodies reacting to the immunized antigen without any additional in vitro screening. An alpaca was immunized with IgG fragments using multiple antigen injections, and the antibody repertoire development was traced via high-throughput sequencing periodically for months. The sequences were processed into clusters, and the antibodies in the 16 most abundant clusters were generated to determine whether the clusters included antigen-binding antibodies. The sequences of most antigen-responsive clusters resembled those of germline cells in the early stages. These sequences were observed to accumulate significant mutations and also showed a continuous sequence turnover throughout the experimental period. The foregoing characteristics gave us >80% successful prediction of clusters composed of antigen-responding VHHs against IgG fragment. Furthermore, when the prediction method was applied to the data from other alpaca immunized with epidermal growth factor receptor, the success rate exceeded 80% as well, confirming the general applicability of the prediction method. Superior to previous studies, we identified the immune-responsive but very rare clusters or sequences from the immunized alpaca without any empirical screening data.

Cellular effects of industrial metal nanoparticles and hydrophilic carbon black dispersion.

The effects of five types of metal nanoparticles, gold (Au), silver (Ag), platinum (Pt), Au-polyvinylpyrrolidone (PVP) colloid, and Pt-PVP colloid, and two types of hydrophilic carbon black on cell behavior were examined. Stable nanoparticle dispersions were prepared and applied to the culture medium of human keratinocyte (HaCaT) and human lung carcinoma (A549) cells for 6 and 24 hr. Then, the mitochondrial activity (MTT assay) and the induction of cellular oxidative stress were examined. The exposure to Au and Ag decreased mitochondrial activity. The exposure to Pt nanoparticles induced an increase in the intracellular reactive oxygen species (ROS) level. In contrast, Au-PVP, Pt-PVP, and hydrophilic carbon black did not exhibit any effects. The observed increase in the ROS level induced by the Pt nanoparticles in this study contradicted our previous findings, in which Pt did not produce chemically reactive molecules. Some nanoparticle dispersions included chemicals as the dispersant, which is used in industrial applications. In some cases, the dispersing agent may have caused some cellular effects. Adsorption of agents on the surface of the nanoparticles may be an important factor here. Hence, the cellular effects of industrial nanoparticles should be evaluated carefully.

Oxidative stress is involved in fatigue induced by overnight deskwork as assessed by increase in plasma tocopherylhydroqinone and hydroxycholesterol.

In this study, we examined the relationship between fatigue and plasma concentrations of antioxidants and lipid peroxidation products. Fourteen healthy volunteers performed overnight desk work for 18h then took a nap for 4h. Participants answered questionnaires of subjective symptoms of fatigue (QSSF) and completed a self-assessment of fatigue using a visual analog scale (VAS). At each test time, they underwent a critical flicker frequency (CFF) test and blood samples were collected. Plasma levels of α-tocopherol (αT) decreased and α-tocopherylquinone (αTQ), the oxidation product of αT, increased. The ratio of 7ß-hydroxycholesterol (7ß-OHCh), the oxidation product of cholesterol, against total cholesterol increased until the end of experiment. αTQ levels correlated with VAS and QSSF scores. The ratio of 7ß-OHCh to total cholesterol and the value of CFF showed a significant correlation. From these results, plasma levels of αTQ and 7ß-OHCh are useful and objective indicators of fatigue induced by overnight deskwork.

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung.

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.

Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area.

Association of cellular influences and physical and chemical properties were examined for 24 kinds of industrial metal oxide nanoparticles: ZnO, CuO, NiO, Sb(2)O(3), CoO, MoO(3), Y(2)O(3), MgO, Gd(2)O(3), SnO(2), WO(3), ZrO(2), Fe(2)O(3), TiO(2), CeO(2), Al(2)O(3), Bi(2)O(3), La(2)O(3), ITO, and cobalt blue pigments. We prepared a stable medium dispersion for each nanoparticle and examined the influence on cell viability and oxidative stress together with physical and chemical characterizations. ZnO, CuO, NiO, MgO, and WO(3) showed a large amount of metal ion release in the culture medium. The cellular influences of these soluble nanoparticles were larger than insoluble nanoparticles. TiO(2), SnO(2), and CeO(2) nanoparticles showed strong protein adsorption ability; however, cellular influences of these nanoparticles were small. The primary particle size and the specific surface area seemed unrelated to cellular influences. Cellular influences of metal oxide nanoparticles depended on the kind and concentrations of released metals in the solution. For insoluble nanoparticles, the adsorption property was involved in cellular influences. The primary particle size and specific surface area of metal oxide nanoparticles did not affect directly cellular influences. In conclusion the most important cytotoxic factor of metal oxide nanoparticles was metal ion release.

Evaluation of cellular influences of platinum nanoparticles by stable medium dispersion.

Platinum nanoparticles have industrial application, for example in catalysis, and are used in consumer products such as cosmetics and supplements. Therefore, among the many nanoparticles, platinum is one of the more accessible nanoparticles for consumers. Most platinum nanoparticles that are used in cosmetics and supplements which have an anti-oxidant activity are modified particles. However, the cellular influences of pristine platinum nanoparticles are still unclear, although it has been reported that platinum nanoparticles induce oxidative stress. In this study, we investigated the cellular influences induced by pure pristine platinum nanoparticles. Platinum nanoparticles of 100% purity were dispersed in a cell culture medium and stable medium dispersion was obtained. The platinum nanoparticle medium dispersion was applied to two kinds of cultured cells, A549 and HaCaT cells, and the cellular influences were examined. Cell viability (MTT assay), cell proliferation (clonogenic assay), apoptosis induction (caspase-3 activity), intracellular ROS level (DCFH assay), and lipid peroxidation level (DPPP assay) were measured as markers of cellular influences. Transmission electron microscope observation showed cellular uptake of platinum nanoparticles. However, the platinum nanoparticles did not drive any markers. It is known that some metal oxide nanoparticles such as NiO and CuO show severe cytotoxicity via metal ion release. Compared with these toxic nanoparticles, the platinum nanoparticles used in this study did not release platinum ions into the culture media. These results suggest that the physically and chemically inactive cellular influences of platinum nanoparticles are small.