Cellular internalization, transcellular transport, and cellular effects of silver nanoparticles in polarized Caco-2 cells following apical or basolateral exposure.

When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticles were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 µg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side.

Protein corona changes mediated by surface modification of amorphous silica nanoparticles suppress acute toxicity and activation of intrinsic coagulation cascade in mice.

Recently, nanomaterial-mediated biological effects have been shown to be governed by the interaction of nanomaterials with some kinds of proteins in biological fluids, and the physical characteristics of the nanomaterials determine the extent and type of their interactions with proteins. Here, we examined the relationships between the surface properties of amorphous silica nanoparticles with diameters of 70 nm (nSP70), their interactions with some proteins in biological fluids, and their toxicity in mice after intravenous administration. The surface modification of nSP70 with amino groups (nSP70-N) prevented acute lethality and abnormal activation of the coagulation cascade found in the nSP70-treated group of mice. Since our previous study showed that coagulation factor XII played a role in the nSP70-mediated abnormal activation of the coagulation cascade, we examined the interaction of nSP70 and nSP70-N with coagulation factor XII. Coagulation factor XII bonded to the surface of nSP70 to a greater extent than that observed for nSP70-N, and consequently more activation of coagulation factor XII was observed for nSP70 than for nSP70-N. Collectively, our results suggest that controlling the interaction of nSP70 with blood coagulation factor XII by modifying the surface properties would help to inhibit the nSP70-mediated abnormal activation of the blood coagulation cascade.

Development of a method of evaluating PuO2 particle diameters using an alpha-particle imaging detector.

It is very important to evaluate the diameters (activity median aerodynamic diameter) of plutonium dioxide (PuO2) particles for internal exposure dose evaluation. In this study, a method of evaluating PuO2 particle diameters using an alpha-particle imaging detector was developed. PuO2 particles with different diameters were modeled by Monte Carlo simulation, and the change in the shape of the energy spectrum for each particle diameter was evaluated. Two different patterns were modeled, namely, the case of 239PuO2 and the case of PuO2 (including isotopic composition of Pu). Multiple regression analysis was performed to determine the PuO2 particle diameter from the obtained parameters. The simulated diameters and the diameters obtained with the regression model were in good agreement. The advantage of using the alpha-particle imaging detector is to measure the alpha energy spectrum for individual particle, and this allows accurate measurement of particle diameter distribution.

Triplet-DNP in magnetically oriented microcrystal arrays.

We explore dynamic nuclear polarization using electron spins in the photo-excited triplet state (Triplet-DNP) in magnetically oriented microcrystal arrays (MOMAs) of pentacene-doped p-terphenyl, in which the individual crystallites are magnetically aligned and UV-cured. In contrast to the conventional approach to Triplet-DNP in powder, which suffers from reduced nuclear polarization due to the averaged electron polarization and the broadening of electron-spin resonance, Triplet-DNP of the MOMAs offers as high dynamic polarization as that attainable in single-crystals. In the case of pentacene-doped p-terphenyl, the enhanced 1H polarization in the one-dimensional MOMA, prepared simply by leaving the suspension in a stationary magnetic field before UV curation, can be higher than that attainable in the powder sample by an order of magnitude and comparable to that in single crystals and in the three-dimensional MOMA made using a modulational rotating field. Triplet-DNP of the MOMAs may find potential applications, such as the polarization of the co-doped target molecules and dissolution experiments.

Air-Bubble-Insensitive Microfluidic Lactate Biosensor for Continuous Monitoring of Lactate in Sweat.

This study aimed to develop a lactate sensor with a microchannel that overcomes the issue of air bubbles interfering with the measurement of lactate levels in sweat and to evaluate its potential for continuous monitoring of lactate in sweat. To achieve continuous monitoring of lactate, a microchannel was used to supply and drain sweat from the electrodes of the lactate sensor. A lactate sensor was then developed with a microchannel that has an area specifically designed to trap air bubbles and prevent them from contacting the electrode. The sensor was evaluated by a person while exercising to test its effectiveness in monitoring lactate in sweat and its correlation with blood lactate levels. Furthermore, the lactate sensor with a microchannel in this study can be worn on the body for a long time and is expected to be used for the continuous monitoring of lactate in sweat. The developed lactate sensor with a microchannel effectively prevented air bubbles from interfering with the measurement of lactate levels in sweat. The sensor showed a concentration correlation ranging from 1 to 50 mM and demonstrated a correlation between lactate in sweat and blood. Additionally, the lactate sensor with a microchannel in this study can be worn on the body for an extended period and is expected to be useful for the continuous monitoring of lactate in sweat, particularly in the fields of medicine and sports.

Distribution and histologic effects of intravenously administered amorphous nanosilica particles in the testes of mice.

Amorphous nanosilica particles (nSP) are being utilized in an increasing number of applications such as medicine, cosmetics, and foods. The reduction of the particle size to the nanoscale not only provides benefits to diverse scientific fields but also poses potential risks. Several reports have described the in vivo and in vitro toxicity of nSP, but few studies have examined their effects on the male reproductive system. The aim of this study was to evaluate the testicular distribution and histologic effects of systemically administered nSP. Mice were injected intravenously with nSP with diameters of 70 nm (nSP70) or conventional microsilica particles with diameters of 300 nm (nSP300) on two consecutive days. The intratesticular distribution of these particles 24h after the second injection was analyzed by transmission electron microscopy. nSP70 were detected within sertoli cells and spermatocytes, including in the nuclei of spermatocytes. No nSP300 were observed in the testis. Next, mice were injected intravenously with 0.4 or 0.8 mg nSP70 every other day for a total of four administrations. Testes were harvested 48 h and 1 week after the last injection and stained with hematoxylin-eosin for histologic analysis. Histologic findings in the testes of nSP70-treated mice did not differ from those of control mice. Taken together, our results suggest that nSP70 can penetrate the blood-testis barrier and the nuclear membranes of spermatocytes without producing apparent testicular injury.

Molecular detection of Blastocystis sp. subtype 14 in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

This study describes the first report of Blastocystis sp. colonization in the sika deer (Cervus nippon) in Japan and in other animals in Hokkaido, Japan. Blastocystis sp. is one of the most widespread intestinal protist in a wide range of animals. Blastocystis sp. isolated from mammalian and avian species have been classified into 17 subtypes (STs). Some of the STs are zoonotic. The aim of this study was to evaluate Blastocystis sp. colonization in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. The Yezo sika deer are currently overabundant and they are expanding their habitat to humans and livestock. A total of 132 deer fecal samples were subjected for molecular detection of Blastocystis sp. Of these, 60 (45.5%) samples were positive using PCR, which targets the small subunit ribosomal RNA gene sequence. All Blastocystis sp. DNA sequences from the Yezo sika deer were genotyped into ST14, which were originally reported in cattle. These findings indicate that the current public health risks of Blastocystis sp. from the Yezo sika deer is low, although more detailed future analysis is required.

Plutonium dioxide particle imaging using a high-resolution alpha imager for radiation protection.

The internal exposure of workers who inhale plutonium dioxide particles in nuclear facilities is a crucial matter for human protection from radiation. To determine the activity median aerodynamic diameter values at the working sites of nuclear facilities in real time, we developed a high-resolution alpha imager using a ZnS(Ag) scintillator sheet, an optical microscope, and an electron-multiplying charge-coupled device camera. Then, we designed and applied a setup to measure a plutonium dioxide particle and identify the locations of the individual alpha particles in real time. Employing a Gaussian fitting, we evaluated the average spatial resolution of the multiple alpha particles was evaluated to be 16.2 ± 2.2 µmFWHM with a zoom range of 5 ×. Also, the spatial resolution for the plutonium dioxide particle was 302.7 ± 4.6 µmFWHM due to the distance between the plutonium dioxide particle and the ZnS(Ag) scintillator. The influence of beta particles was negligible, and alpha particles were discernible in the alpha-beta particle contamination. The equivalent volume diameter of the plutonium dioxide particle was calculated from the measured count rate. These results indicate that the developed alpha imager is effective in the plutonium dioxide particle measurements at the working sites of nuclear facilities for internal exposure dose evaluation.

Urban aerosols induce pro-inflammatory cytokine production in macrophages and cause airway inflammation in vivo.

Urban air pollution, especially in developing countries, is a crucial environmental problem. Urban aerosols may contain various kinds of substances and induce harmful effects such as allergic diseases. Therefore, it is critical to clarify the biological effects of urban aerosols on human health. In this study, we evaluated the induction of airway inflammation in vitro and in vivo due to exposure of urban aerosols. We investigated cytokine production and nuclear factor-kappaB (NF-kappaB) activation after stimulation of macrophage cells by exposure of urban aerosols. Urban aerosols were found to induce the production of interleukin (IL)-8, tumor necrosis factor-alpha and IL-1beta on macrophage cells. In addition, we showed that NF-kappaB pathway regulated the urban aerosols-induced inflammatory cytokine response. Moreover, the intranasal administration of urban aerosols resulted in increases in the total cell number in bronchoalveolar lavage and infiltration of eosinophils in lung tissue. These results indicate that urban aerosols induce respiratory inflammation and onset of inflammatory disease due to an activation of the immune system.

A REMOTE CONTINUOUS AIR MONITORING SYSTEM FOR MEASURING AIRBORNE ALPHA CONTAMINATION.

We developed a remote continuous air monitoring (RCAM) system. The RCAM system consisted of a personal air monitor and a robot. The personal air monitor (poCAMon, SARAD, Germany) had a 400-mm2 ion-injected silicon detector and a membrane air filter with 25 mmφ. The personal air monitor provides the alpha energy spectra for any measurement time interval. Demonstration measurements were taken underground at the Mizunami Underground Research Laboratory and at a poorly ventilated concrete building. The RCAM system was remotely operated and successfully measured the 222Rn progeny even though the relative humidity was almost 100%. In the measured alpha spectra, the peaks of 218Po (6.0-MeV alpha) and 214Po (7.7-MeV alpha) were clearly identified. Our developed monitor is promising for alpha dust monitoring in a high gamma-ray environment or contaminated areas where a worker cannot safely physically enter.

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido, Japan.

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.

Detection of alpha particle emitters originating from nuclear fuel inside reactor building of Fukushima Daiichi Nuclear Power Plant.

We measured alpha emitters obtained from a reactor building in the Fukushima Daiichi Nuclear Power Plant (FDNPP) by using an alpha particle imaging detector. For developing the detector, we used a very thin (0.05-mm-thick) a cerium-doped Gd3(Ga,Al)5O12 (Ce:GAGG) scintillator and silicon photomultiplier (SiPM) arrays as the photodetector. The floor of the reactor building in FDNPP was wiped off by using smear papers, and the radioactivity of these papers was measured by the alpha particle imaging detector. In addition, we measured a Plutonium (Pu) sample (mainly 5.5 MeV alpha particles from 238Pu) obtained from a nuclear fuel facility by using of the same detector for comparison with the smear papers. The alpha spectrum was in the energy range of 5-6 MeV, which corresponds to the alpha particle energy of 238Pu (5.5 MeV). The correlation coefficient of the alpha spectra of the smear papers and the Pu sample had a strong positive linear relation. Moreover, the peak of 241Am was identified by gamma spectrum measurement. Based on these results, we report actual findings of alpha emitters in the FDNPP reactor buildings originating from nuclear fuels. The surface contamination level of alpha emitters exceeded 4 Bq/cm2.

A biological study establishing the endotoxin limit for osteoblast and adipocyte differentiation of human mesenchymal stem cells.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions, such as hypoxia or inflammation after transplantation in vivo. Although endotoxin testing is required for evaluating the quality and safety of transplanted MSCs, no reports on their dose response to endotoxins are available to establish the limits for in vitro MSC culture systems. In the present study, we aimed to accurately quantify the risk of endotoxin contamination in cell culture systems to establish an acceptable endotoxin limit for the differentiation of hMSC osteoblasts and adipocytes. METHODS: Three types of bone marrow-derived hMSCs (hMSC-1: 21-year-old, M/B; hMSC-2: 36-year-old, M/B; hMSC-3: 43-year-old, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in osteogenic or adipogenic differentiation media, respectively, from commercial kits, containing various concentrations of endotoxin (0.01-100 ng/ml). The degree of adipocyte and osteoblast differentiation was estimated by fluorescent staining of lipid droplets and hydroxyapatite, respectively. To clarify the molecular mechanism underlying the effect of endotoxin on hMSC differentiation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. RESULTS: Although endotoxin did not effect the adipocyte differentiation of hMSCs, osteoblast differentiation was enhanced by various endotoxin concentrations: over 1 ng/ml, for hMSC-1; 10 ng/ml, for hMSC-2; and 100 ng/ml, for hMSC-3. Proteomic analysis of hMSC-1 cells revealed up-regulation of many proteins related to bone formation. These results suggested that endotoxin enhances the osteoblast differentiation of MSCs depending on the cell type. CONCLUSIONS: Since endotoxins can affect various cellular functions, an endotoxin limit should be established for in vitro MSC cultures. Its no-observed-adverse-effect level was 0.1 ng/ml based on the effect on the hMSC osteoblast differentiation, but it may not necessarily be the limit for ADSCs.

DEVELOPMENT OF A NEW DETECTOR SYSTEM TO EVALUATE POSITION AND ACTIVITY OF PLUTONIUM PARTICLES IN NASAL CAVITIES.

Plutonium dioxide (PuO2) is used to fabricate a mixed oxide fuel for fast breeder reactors. When a glove box containing PuO2 fails, such as by rupture of a glove or a vinyl bag, airborne contamination of plutonium (Pu) can occur. If a worker inhales PuO2 particles, they will be continually irradiating their lung tissue with alpha particles, and this could cause lung cancer. The nasal smear and nose blow methods are useful for checking workers for PuO2 intake in the field. However, neither method can evaluate the quantitative activity of Pu. No alpha-particle detector that can be used for direct measurements in the nasal cavity has been developed. For direct and quantitative measurement, it is required that a shape of the detector should be a fine bar which inserts itself in the nose to measure the accurate activity of Pu. Therefore, we developed a nasal monitor capable of directly measuring the activity of Pu in the nasal cavity to estimate the internal exposure dose of a worker. Prismatic-shaped 2 × 2 acrylic light guides were used to compose a detector block, and a ZnS(Ag) scintillator was adhered to the surface of these light guides. Silicon photomultiplier (SiPM) arrays with 8 × 8 channels were used as a photodetector. Actual PuO2 particles were measured using the nasal monitor. The nasal monitor could be directly inserted in the nasal cavities, and the activity distribution of Pu was obtained by the nasal monitor. The average efficiencies in 4-pi were 11.4 and 11.6% for the left and right nasal cavities, respectively. The influence of gamma and beta rays from Cesium-137 (137Cs) Strontium-90 (90Sr) on the detection of the alpha particles of Pu was negligible. The difference in the measured Pu activity between the ZnS(Ag) scintillation counter and the nasal monitor was within 4.0%. Therefore, it was considered that the developed nasal monitor could be used in direct Pu determination to estimate the internal exposure dose of workers.

Cetuximab retreatment in patients with metastatic colorectal cancer who exhibited a clinical benefit in response to prior cetuximab: A retrospective study.

Clinical benefits of cetuximab retreatment in patients with metastatic colorectal (mCRC) have been reported. In the present study, the effect of cetuximab retreatment on predictive markers was investigated by evaluating the clinical benefit of initial cetuximab treatment prior to cetuximab retreatment. Between November 2012 and March 2017, 14 patients with KRAS proto-oncogene GTPase exon 2 wild-type mCRC who exhibited a clinical benefit (confirmed stable disease for at least 6 months or a clinical response) to an initial cetuximab-based regimen, who received multiple lines of chemotherapy following disease progression and ultimately received a second cetuximab and irinotecan regimen, were retrospectively analyzed. For retreatment, patients received bi-weekly irinotecan (120-150 mg/m2) combined with cetuximab (400 mg/m2 as an initial dose, followed by 250 mg/m2, weekly). The median age of the 14 patients (11 males, 3 females) was 68 years (32-77). The median progression-free survival (PFS) following prior cetuximab-based therapy was 6.6 months (range, 4.1-18.4). Initial cetuximab treatment was administered as a first-line treatment in 11 patients, a second-line treatment in 1 patient and a third-line treatment in 2 patients. The median interval time between the last cycle of initial cetuximab-based therapy and the first cycle of cetuximab retreatment was 13.1 months (range, 6.0-37.1). The objective response rate of cetuximab retreatment was 21.4% and the median PFS was 4.4 months (95% confidence interval, 1.4-5.6). The Spearman's correlation coefficient for the PFS following retreatment and duration of initial cetuximab-based regimens demonstrated a more marked correlation compared with that between the PFS following retreatment and the interval time between the two regimens (r=0.45, P=0.11 vs. r=0.08, P=0.79). Cetuximab retreatment may provide clinical benefit to patients with mCRC who were good responders with longer periods of initial cetuximab-based therapy.

Proof of concept testing of a positive reference material for in vivo and in vitro skin irritation testing.

In vivo and in vitro irritation testing is important for evaluating the biological safety of medical devices. Here, the performance of positive reference materials for skin irritation testing was evaluated. Four reference standards, referred to as Y-series materials, were analyzed: a polyvinyl chloride (PVC) sheet spiked with 0 (Y-1), 1.0 (Y-2), 1.5 (Y-3), or 10 (Y-4) parts of Genapol X-080 per 100 parts of PVC by weight. Y-1, Y-2, and Y-3 did not induce skin irritation responses in an in vitro reconstructed human epidermis (RhE) tissue model, as measured by tissue viability or interleukin-1α release, or in an in vivo intracutaneous response test using rabbits. In contrast, Y-4 extracts prepared with saline or sesame oil at 37°C and 50°C clearly elicited positive irritation responses, including reduced viability (< 50%) and significantly higher interleukin-1α release compared with the solvent alone group, in the RhE tissue model and an intracutaneous response test, where substantial necrosis was observed by histopathology. The positive skin irritation responses induced in vitro under various extraction conditions, as well as those elicited in vivo, indicate that Y-4 is an effective extractable positive control material for in vivo and in vitro skin irritation tests of medical devices. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2807-2814, 2018.

Alternative plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester, for blood containers with protective effects on red blood cells and improved cold resistance.

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC), is eluted from PVC-made blood containers and protects against red blood cell (RBC) hemolysis. However, concerns have arisen regarding the reproductive and developmental risks of DEHP in humans, and the use of alternative plasticizers for medical devices has been recommended worldwide. In this study, we propose that the use of a novel plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH), could help produce more useful and safe blood containers. PVC sheet containing DL9TH and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH) provides comparable or superior protective effects to RBCs relative to PVC sheet containing DEHP or di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH® , an alternative plasticizer that has been used in PVC sheets for blood containers). The total amount of plasticizer eluted from DOTH/DL9TH-PVC sheets is nearly the same as that eluted from DEHP-PVC sheets. In addition, DOTH/DL9TH-PVC has better cold resistance than DEHP- and DINCH® -PVC sheets. In vitro and in vivo tests for biological safety based on International Organization for Standardization guidelines (10993 series) suggest that the DOTH/DL9TH-PVC sheet can be used safely. Subchronic toxicity testing of DL9TH in male rats in accordance with the principles of Organisation for Economic Co-operation and Development Test Guideline 408 showed that DL9TH did not induce adverse effects up to the highest dose level tested (717 mg/kg body weight/day). There were no effects on testicular histopathology and sperm counts, and no indications of endocrine effects: testosterone, thyroid-stimulating hormone, follicle-stimulating hormone, and 17ß-estradiol were unchanged by the treatment, compared with the control group. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1052-1063, 2018.

A biological study establishing the endotoxin limit for in vitro proliferation of human mesenchymal stem cells.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions such as hypoxia or inflammation after in vivo transplantation. Although endotoxin testing is a requirement for evaluating the quality and safety of transplanted MSCs, there have been no reports on the dose response to endotoxins to establish limits for in vitro MSC culture systems. The present study aimed to accurately quantify the risk of endotoxin contamination in cell culture systems in order to establish the acceptable endotoxin limit for hMSC proliferation. METHODS: Three types of bone marrow-derived hMSC (hMSC-1: 21 years, M/B; hMSC-2: 36 years, M/B; hMSC-3: 43 years, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in medium from commercial kits containing various concentrations of endotoxin (0.1-1000 ng/ml). The proliferative capacity of cells was estimated by cell counts using a hemocytometer. To clarify the molecular mechanism underlying the effect of endotoxin on hMSCs proliferation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. The expression of Cu/Zn-type superoxide dismutase (SOD1) and Fe/Mn-type superoxide dismutase (SOD2) induced in hMSCs by endotoxin stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), and the effect of SOD2 on hMSC proliferation was also estimated. RESULTS: Although there was no change in cell morphology during the culture period, proliferative capacity increased with endotoxin concentration to over 0.1 ng/ml for ADSCs, 1 ng/ml for hMSC-1, and 100 ng/ml for hMSC-2; hMSC-3 proliferation was unaffected by the presence of endotoxin. A proteomic analysis of hMSC-1 revealed that various proteins related to the cell cycle, apoptosis, and host defense against infection were altered by endotoxin stimulation, whereas SOD2 expression was significantly and consistently upregulated during the culture period. The latter was also confirmed by ELISA. Moreover, recombinant SOD2 increased proliferative capacity in hMSC-1 cells in a manner similar to endotoxin. These results suggest that endotoxin protects MSCs from oxidative stress via upregulation of SOD2 to improve cell survival. CONCLUSIONS: Since endotoxins can affect various cellular functions, an endotoxin limit should be set for in vitro MSC cultures. The lowest observed adverse effect level was determined to be 0.1 ng/ml based on the effect on MSC proliferation.

Pilot study on novel blood containers with alternative plasticizers for red cell concentrate storage.

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC) blood containers, is eluted from the blood containers and exerts protective effects on red blood cells. However, a concern for detrimental effects of DEHP on human health has led to the development of potential DEHP substitutes. Here, we compared the red blood cell preservation ability of two types of non-DEHP blood containers with safe alternative plasticizers to that of DEHP blood containers. Red cell concentrates in mannitol-adenine-phosphate solution (MAP/RCC) were stored for 6 weeks in PVC blood bags containing DEHP, di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH) and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH), or 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH) and DOTH. There was no significant difference in the total amount of plasticizer eluted into MAP/RCC (till 3 weeks from the beginning of the experiment), hemolysis of MAP/RCC, and osmotic fragility of MAP/RCC between the non-DEHP blood containers and DEHP blood containers. Hematological and blood chemical indices of MAP/RCC in all containers were nearly the same. Thus, DOTH/DINCH and DOTH/DL9TH blood containers demonstrate the same quality of MAP/RCC storing as the DEHP blood containers. Since DOTH, DINCH, and DL9TH were reported to be safe, DOTH/DINCH and DOTH/DL9TH blood containers are promising candidate substitutes for DEHP blood containers.