Performance evaluation of bactericidal effect and endotoxin inactivation by low-temperature ozone/hydrogen peroxide mixed gas exposure.

This study evaluated the performance of a new O3 /H2 O2 mixed gas sterilization instrument for killing microorganisms and inactivating bacterial endotoxin at low temperatures. Sterility assurance level was achieved by an over 6-log reduction of Geobacillus stearothermophilus ATCC 12980, and the decimal reduction value was 0.77 min in sterilization mode. A reduction of over 3 logs in Limulus amebocyte lysate coagulation activity of purified endotoxin from Escherichia coli was observed after treatment in endotoxin-inactivation mode. The same inactivation ability was observed when treating dried bacterial cells. Biomaterials made of polymer or metal did not exhibit cytotoxicity after gas exposure at O3 concentrations below 200 ppm. As the results of human cell-based pyrogen testing, significant amounts of endotoxin that were over the limit for medical devices contacting cerebrospinal fluid (2.15 EU/device) were detected on scissors washed with a washer-disinfector and sterilized with ethylene oxide or autoclaving. In contrast, endotoxin decreased to 0.29 ± 0.05 EU/device after O3 /H2 O2 mixed gas sterilization in endotoxin-inactivation mode. Compared to conventional gas sterilization methods, O3 /H2 O2 mixed gas has high sterilization ability and a strong capacity to inactivate endotoxin. It is expected that this sterilization technology will improve the safety of reusable medical devices and utensils for regenerative medicine.

A novel method to eliminate the influence of absorbed lipids on the characterization of ultra-high molecular weight polyethylene using Fourier-transform infrared spectroscopy.

BACKGROUND: Fourier-transform infrared spectroscopy (FTIR) is one of the standard methods to analyze ultra-high molecular weight polyethylene (UHMWPE) in orthopedic implants. For retrieved components, lipid extraction using an organic solvent prior to the measurement is necessary to eliminate the influence of lipids absorbed in vivo. However, its influence on the measurement has not been substantially investigated. OBJECTIVE: To investigate the influence of lipid extraction on the FTIR analysis of UHMWPE and to develop a novel method to obtain reliable results without inconvenient lipid extraction. METHODS: FTIR analysis was repeatedly performed on UHMWPE specimens from retrieved components before and after lipid extraction under various conditions. A method to calculate the extent of influence of the absorbed lipids from the FTIR spectra was developed using a peak separation technique. RESULTS: An elevated temperature was necessary for lipid extraction; however, it had the potential to influence the results if the conditions were not properly controlled. The results obtained using the peak separation technique coincided with those obtained after lipid extraction. CONCLUSION: The use of the peak separation technique enables the efficient acquisition of reliable results without the need for lipid extraction.

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.

Conjugation of two RNA aptamers improves binding affinity to AML1 Runt domain.

To develop a high-affinity aptamer against AML1 Runt domain, two aptamers were conjugated based on their structural information. The newly designed aptamer Apt14 was generated by the conjugation of two RNA aptamers (Apt1 and Apt4) obtained by SELEX against AML1 Runt domain, resulting in improvement in its binding performance. The residues of AML1 Runt domain in contact with Apt14 were predicted in silico and confirmed by mutation and NMR analyses. It was suggested that the conjugated internal loop renders additional contacts and is responsible for the enhancement in the binding affinity. Conjugation of two aptamers that bind to different sites of the target protein is a facile and robust strategy to develop an aptamer with higher performance.

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.

[Comprehensive analyses of hydrolyzed wheat protein using shotgun proteomics].

Hydrolyzed wheat protein (HWP; hydrolyzed gluten) is used in various types of products worldwide. Several cases of wheat-dependent, exercise-induced anaphylaxis following exposure to HWP (Glupearl 19S) in cosmetics have been reported. Glupearl 19S was produced from the gluten after partial hydrolysis with hydrogen chloride, and its allergenicity is larger than that of gluten (Adachi R., Allergy 2012;67:1392-9.). It is considered that provocation of allergic manifestations is caused by deamidated gluten in food and/or non-food products. Moreover, an increasing number of studies have shown that HWP can induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. However, the essential molecular properties and profiles of HWP are still unknown. In this study, bioinformatic and multivariate analyses using shotgun proteomics have revealed that 27 proteins significantly decreased in Glupearl 19S compared with intact gluten as shown by the ratio of ion signal intensity of tryptic peptides. In contrast, a single protein significantly increased in HWP compared with intact gluten as shown by the ratio of ion signal intensity of tryptic peptides. Furthermore, we have identified six Glupearl 19S-specific peptides using shotgun proteomics, database searches on Mascot Sequence Query, and de novo sequencing. The six peptides were identified as the specific markers of Glupearl 19S.

Calcium-incorporated titanium surfaces influence the osteogenic differentiation of human mesenchymal stem cells.

In this study, a titanium surface was chemically modified with calcium ions and assessed for its influence on osteogenic differentiation and molecular responses of human mesenchymal stem cells (hMSCs). Titanium disks were treated with NaOH (NaOH treatment), NaOH + CaCl2 (CaCl2 treatment), or NaOH + Ca(OH)2 (Ca(OH)2 treatment). Ca(OH)2 treatment caused significantly greater calcium incorporation onto the titanium surface and apatite formation than CaCl2 treatment. The morphology of hMSCs differed on CaCl2- and Ca(OH)2-treated disks. The osteopontin (OPN) expression in hMSCs cultured on CaCl2-treated titanium was significantly higher than that in cells cultured on NaOH-treated disks; OPN expression was significantly higher in cells cultured on Ca(OH)2-treated disks than on un-, NaOH-, and CaCl2-treated disks. Osteocalcin (OCN) protein expression in hMSCs cultured on Ca(OH)2-treated disks was significantly higher than that on all the other disks. Comparative expression profiling by DNA microarray and pathway analyses revealed that calcium modification of the titanium surface induced integrin ß3 after OPN upregulation and promoted Wnt/ß-catenin signaling in hMSCs. In addition, Ca(OH)2 treatment upregulated the expression of bone morphogenetic protein 2, cyclooxygenase 2, and parathyroid hormone-like hormone in comparison to CaCl2 treatment. These observations suggest that calcium-modified titanium surfaces affect osteogenic differentiation in hMSCs and that Ca(OH)2 treatment induced osteogenic differentiation in hMSCs, whereas CaCl2 treatment had a limited effect.

Anti-HIV and immunomodulation activities of cacao mass lignin-carbohydrate complex.

BACKGROUND: Recently, a prominent antiviral and macrophage stimulatory activity of cacao lignin-carbohydrate complex (LCC) has been reported. However, the solubility and sterility of LCC have not been considered yet. In the present study, complete solubilisation and sterilisation was achieved by autoclaving under mild alkaline conditions and the previously reported biological activities were re-examined. MATERIALS AND METHODS: LCCs were obtained by 1% NaOH extraction and acid precipitation, and a repeated extraction-precipitation cycle. Nitric oxide (NO) and cytokine productions were assayed by the Griess method and ELISA, respectively. Inducible NO synthase (iNOS) expression was determined by Western blot analysis. Superoxide anion, hydroxyl radical and nitric oxide radical-scavenging activity was determined by ESR spectroscopy. RESULTS: Cacao mass LCC showed reproducibly higher anti-HIV activity than cacao husk LCC. Cacao mass LCC, up to 62.5 µg/ml, did not stimulate mouse macrophage-like cells (RAW264.7 and J774.1) to produce NO, nor did it induce iNOS protein, in contrast to lipopolysaccharide (LPS). Cacao mass LCC and LPS synergistically stimulated iNOS protein expression, suggesting a different point of action. Cacao mass LCC induced tumour necrosis factor-α production markedly less than LPS, and did not induce interleukin-1ß, interferon-α or interferon-γ. ESR spectroscopy showed that cacao mass LCC, but not LPS, scavenged NO produced from NOC-7. CONCLUSION: This study demonstrated several new biological activities of LCCs distinct from LPS and further confirmed the promising antiviral and immunomodulating activities of LCCs.

Development of an in vitro screening method for safety evaluation of nanomaterials.

To evaluate the role of particle size in cytotoxicity tests of nanomaterials (NMs), we exposed Chinese hamster cells to polystyrene (PS) spheres with defined diameters ranging from 0.1 to 9.2 microm. We found that the 4.45-microm PS particles were most cytotoxic while sizes 0.1 and 0.2 microm showed no cytotoxicity up to 1000 microg/ml. In the chromosome aberration test, the 4.45-microm PS particles induced polyploidy in a mass concentration-dependent manner in 24- and 48-h treatments. The 5.26-microm PS particles induced polyploidy only at 1000 microg/ml for 48 h. Next, we performed the cytotoxicity test with as-grown single walled carbon nanohorns (NHas). These were suspended in DMSO and then transferred into the culture medium followed by sonication. Six suspensions differently sonicated showed the same apparent toxicity, although the total particle size distributions differed. However, the sizes of NHas particles predicted to be most toxic from the experiments with PS particles, i.e. 1.01-4.47 microm constituted 40-60% of all particles in all six suspensions. The results suggest that the cytotoxicity of NMs in suspension depends on specific sizes of aggregates and therefore suspensions should be checked with regard to particle size distributions in assays of toxic effects. The uptake of particles into cells was confirmed by confocal microscopy.

Endotoxic properties of lipid A from Comamonas testosteroni.

The lipid A from Comamonas testosteroni has been isolated and its complete chemical structure determined [Iida, T., Haishima, Y., Tanaka, A., Nishijima, K., Saito, S. & Tanamoto, K. (1996). Eur J Biochem 237, 468-475]. In this work, the relationship between its chemical structure and biological activity was studied. The lipid A was highly homogeneous chemically and was characterized by the relatively short chain length (C(10)) of the 3-hydroxy fatty acid components directly bound to the glucosamine disaccharide backbone by either amide or ester linkages. The lipid A exhibited endotoxic activity in all of the assay systems tested (mitogenicity in mouse spleen cells; induction of tumour necrosis factor alpha release from both mouse peritoneal macrophages and mouse macrophage-like cell line J774-1, as well as from the human monocytic cell line THP-1; induction of nitric oxide release from J774-1 cells; Limulus gelation activity and lethal toxicity in galactosamine-sensitized mice) to the same extent as did 'Salmonella minnesota' lipid A or Escherichia coli LPS used as controls. The strong endotoxic activity of the C. testosteroni lipid A indicates that the composition of 3-hydroxydecanoic acid is not responsible for the low endotoxicity of the lipid A observed in members of the genus Rhodopseudomonas, as has previously been suggested. Furthermore, both the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3' position, and the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2, do not affect the manifestation of endotoxic activity or species specificity.

Endotoxic properties of free lipid A from Porphyromonas gingivalis.

The relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098-2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.