The effects of continuous exposure to low-dose chlorine dioxide gas on the characteristics of induced pluripotent stem cells.

Background: Recently, various regenerative therapies have been developed based on induced pluripotent stem (iPS) cells. However, hygienic control strategies have not been established at the manufacturing facilities. We aimed to evaluate the safety and effects of continuous exposure to low-dose chlorine dioxide (ClO2) gas on cell fates, and to determine the optimum dose for safe usage of this disinfectant. Methods: We cultured an iPS cell line in the absence or presence of various doses of ClO2 gas. We evaluated cell proliferation, cell death, the maintenance of undifferentiated state, and cell senescence. Results: We found that iPS cell proliferation was not affected by 0.05 or 0.1 ppmv ClO2 gas in the atmosphere. Although 0.1 ppmv ClO2 slightly affected apoptosis, it was not a significant effect. Moreover, neither at 0.05 nor 0.1 ppmv ClO2 gas significantly affected the characteristics of iPS cells. Discussion and conclusion: Continuous exposure to 0.05 or 0.1 ppmv ClO2 gas did not affect the fate of iPS cells. These results may contribute to the development of new strategies for hygiene control in cell processing facilities.

Effects of continuous exposure to low concentration of ClO2 gas on the growth, viability, and maintenance of undifferentiated MSCs in long-term cultures.

INTRODUCTION: Hygienic management is more important in the manufacturing of cell products than in the production of chemical agents, because cell material and final product cannot be decontaminated. On the other hand, especially in the selection of hygienic agent, the adverse effects on the cells must be considered as well as the decontamination effect. ClO2 is a potent disinfectant, which is now expected as a safe and effective hygienic agent in the field of cell production. In this study, we investigated the effects of low dose ClO2 gas in the atmosphere of CO2 incubator on the characteristics of MSCs cultured in it. METHODS: First, we installed a ClO2 generator to a CO2 incubator for cell culture in which a constant level of ClO2 can be maintained. After culturing human cord derived MSCs in the CO2 incubator, the characteristics of cells were analyzed. RESULTS: Continuous exposure to 0.05 ppmv of ClO2 gas did not affect cell proliferation until at least 8th passage. In the FACS analysis, antigens usually expressed on MSCs, CD105, CD90, CD44, CD73 and CD29, were positively observed, but differentiation markers, CD11b and CD34, were little expressed on the MSCs exposed to 0.05 ppmv or 0.1 ppmv of ClO2 gas just as on the control cells. Also in the investigation for cell death, 0.05 ppmv and 0.1 ppmv of ClO2 gas little affected the viability, apoptosis or necrosis of MSCs. Furthermore, we assessed senescence using SA-ß-gal staining. Although the frequency of stained cells cultured in 0.1 ppmv of ClO2 gas was significantly increased than that of not exposed cells, the stained cells in 0.05 ppmv were rare and their frequency was almost the same as that in control. CONCLUSIONS: All these results indicate that, although excessive concentration of ClO2 gas induces senescence but neither apoptosis nor cell differentiation, exposure to 0.05 ppmv of ClO2 gas little affected the characteristics of MSCs. In this study we demonstrate that continuous exposure to appropriate dose of ClO2 gas can be safely used as decontamination agent in cell processing facilities.

Generation and Measurement of Chlorine Dioxide Gas at Extremely Low Concentrations in a Living Room: Implications for Preventing Airborne Microbial Infectious Diseases.

BACKGROUND/AIMS: Preventing respiratory diseases caused by airborne microbes in enclosed spaces is still not satisfactorily controlled. At extremely low concentrations (about 30 parts per billion), chlorine dioxide (ClO2) gas can inactivate airborne microbes and prevent respiratory disease. It has no toxic effect on animals at this level. However, controversies still remain regarding how to measure concentrations of ClO2 gas at such low levels. It is therefore necessary to prove that measured gas concentrations are accurate and reproducible. METHODS: ClO2 gas was released from a gas generator and its concentration was measured by a novel highly sensitive gas analyzer. We compared its data with those from ion chromatography. RESULTS: We demonstrate that the gas concentrations measured in a room using the gas analyzer are accurate and reproducible after comparing the results with those from ion chromatography. However, the temperature dependence of the gas analyzer was found. Therefore, data correction is required for each temperature at which gas concentration is measured. A theoretical analysis of the gas concentrations predicted by the rate of ClO2 gas released from the ClO2 generator was also performed. CONCLUSION: Our results advance progress toward using low concentration ClO2 gas to prevent airborne infectious diseases such as influenza.

Inactivation of Airborne Bacteria and Viruses Using Extremely Low Concentrations of Chlorine Dioxide Gas.

Infectious airborne microbes, including many pathological microbes that cause respiratory infections, are commonly found in medical facilities and constitute a serious threat to human health. Thus, an effective method for reducing the number of microbes floating in the air will aid in the minimization of the incidence of respiratory infectious diseases. Here, we demonstrate that chlorine dioxide (ClO2) gas at extremely low concentrations, which has no detrimental effects on human health, elicits a strong effect to inactivate bacteria and viruses and significantly reduces the number of viable airborne microbes in a hospital operating room. In one set of experiments, a suspension of Staphylococcus aureus, bacteriophage MS2, and bacteriophage ΦX174 were released into an exposure chamber. When ClO2 gas at 0.01 or 0.02 parts per million (ppm, volume/volume) was present in the chamber, the numbers of surviving microbes in the air were markedly reduced after 120 min. The reductions were markedly greater than the natural reductions of the microbes in the chamber. In another experiment, the numbers of viable airborne bacteria in the operating room of a hospital collected over a 24-hour period in the presence or absence of 0.03 ppm ClO2 gas were found to be 10.9 ± 6.7 and 66.8 ± 31.2 colony-forming units/m3 (n = 9, p < 0.001), respectively. Taken together, we conclude that ClO2 gas at extremely low concentrations (≤0.03 ppm) can reduce the number of viable microbes floating in the air in a room. These results strongly support the potential use of ClO2 gas at a non-toxic level to reduce infections caused by the inhalation of pathogenic microbes in nursing homes and medical facilities.

CH-π interaction in VQIVYK sequence elucidated by NMR spectroscopy is essential for PHF formation of tau.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. Investigation of the mechanism of tau aggregation is important for the clarifying the cause of AD and the development of therapeutic drugs. The microtubule-binding domain, which consists of repeats of similar amino acids (R1-R4) is thought to form the core component of paired helical filament (PHF). The hexapeptide(306) VQIVYK(311) of R3 has been shown to take a key role of promoting tau aggregation and assumed that its CH-π interaction between the side chains of Ile308 and Tyr310 would contribute in stabilizing the filament. In this work, we investigated a short isoform of tau (4RTau), R3, VQIVYK peptide and their mutants by thioflavin S (ThS) fluorescence, and NMR measurements, and proved for the first time that this CH-π interaction stabilizes the filament at the atomic level. In addition, by molecular modeling, we revealed that this interaction further supports an extended amphipathic structure for molecular self-association during the process of PHF formation of tau protein. The present work indicates new approach that inhibits the CH-π interaction for developing a therapeutic agent for AD.

C-H ... π interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau.

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of ß-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.