A mixed antioxidant supplement improves cognitive function, and coordination in aged mice.

Accumulation of oxidative damage increases the risk of several disorders. To prevent these diseases, people consume supplements. However, there is little evidence of the impact of supplement intake on cognitive function. Recently, frailty and sarcopenia have become serious issues, and these phenomena include a risk of mild cognitive impairment. In this study, aged mice were fed the combination supplement and cognitive and motor functions were measured. Following 1 month of treatment with the supplement, significant improvements in cognitive function and neuromuscular coordination were observed. Following 2 weeks of treadmill training, treatment with the supplement dramatically increased running distance compared to that in untreated normal aged mice. Serum indices such as triglyceride and total cholesterol were significantly decreased in the supplement-treated aged mice compared to untreated aged mice. These results indicate that the combination supplement may play a role in maintaining cognitive function, coordination ability and improving lipid metabolism.

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud's phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer's disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.

Electrolyzed hypochlorous acid water exhibits potent disinfectant activity against various viruses through irreversible protein aggregation.

It is essential to employ efficient measures to prevent the transmission of pathogenic agents during a pandemic. One such method involves using hypochlorous acid (HClO) solution. The oxidative properties of HClO water (HAW) can contribute to its ability to eliminate viral particles. Here, we examined a highly purified slightly acidic hypochlorous acid water (Hp-SA-HAW) obtained from the reverse osmosis membrane treatment of an electrolytically-generated SA-HAW for its anti-viral activity and mode of action on viral proteins. Hp-SA-HAW exhibited broad-spectrum antiviral effects against various viruses, including adenovirus, hepatitis B virus, Japanese encephalitis virus (JEV), and rotavirus. Additionally, Hp-SA-HAW treatment dose-dependently resulted in irreversibly aggregated multimers of the JEV envelope and capsid proteins. However, Hp-SA-HAW treatment had no discernible effect on viral RNA, indicating that Hp-SA-HAW acts against amino acids rather than nucleic acids. Furthermore, Hp-SA-HAW substantially reduced the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the ancestral variant and other multiple variants. Hp-SA-HAW treatment induced the aggregation of the SARS-CoV-2 spike and nuclear proteins and disrupted the binding of the purified spike protein of SARS-CoV-2 to human ACE2. This study demonstrates that the broad-spectrum virucidal activity of highly purified HClO is attributed to viral protein aggregation of virion via protein oxidation.

Transplantation of Chemical Compound-Induced Cells from Human Fibroblasts Improves Locomotor Recovery in a Spinal Cord Injury Rat Model.

The development of regenerative medicine using cell therapy is eagerly awaited for diseases such as spinal cord injury (SCI), for which there has been no radical cure. We previously reported the direct conversion of human fibroblasts into neuronal-like cells using only chemical compounds; however, it is unclear whether chemical compound-induced neuronal-like (CiN) cells are clinically functional. In this study, we partially modified the method of inducing CiN cells (termed immature CiN cells) and examined their therapeutic efficacy, in a rat model of SCI, to investigate whether immature CiN cells are promising for clinical applications. Motor function recovery, after SCI, was assessed using the Basso, Beattie, and Bresnahan (BBB) test, as well as the CatWalk analysis. We found that locomotor recovery, after SCI in the immature CiN cell-transplanted group, was partially improved compared to that in the control group. Consistent with these results, magnetic resonance imaging (MRI) and histopathological analyses revealed that nerve recovery or preservation improved in the immature CiN cell-transplanted group. Furthermore, transcriptome analysis revealed that immature CiN cells highly express hepatocyte growth factor (HGF), which has recently been shown to be a promising therapeutic agent against SCI. Our findings suggest that immature CiN cells may provide an alternative strategy for the regenerative therapy of SCI.

Twendee X, a mixed antioxidant supplement, improves cognitive function, coordination, and neurotrophic factor expression in long-term vitamin E-deficient mice.

Oxidation products gradually accumulate during senescence, enhancing the risk of onset of many severe diseases. One such disease is dementia, and the number of cases of dementia, including Alzheimer's disease, has been increasing world-wide. These diseases can be prevented via attenuation of age-related physiological dysfunction; one preventive approach is the ingestion of antioxidants such as vitamin C and vitamin E. Many antioxidants are readily available commercially. Ingestion of mixed antioxidants is expected to provide further beneficial effects for human health. In this study, we used vitamin E-deficient mice as an animal model of increased oxidative stress and assessed the effects of dosing with mixed antioxidants. Administration of a commercial mixed antioxidant formula, Twendee X significantly improved cognitive function and coordination compared to untreated vitamin E-deficient animals. Furthermore, the levels of brain-derived neurotrophic factor and nerve growth factor were significantly increased in the cerebral cortex of Twendee X-dosed vitamin E-deficient mice compared to untreated animals. These results indicate that intake of a mixed antioxidant supplement may be beneficial to human health, even after oxidative stress has begun. In the next stage, it will be necessary to compare with other antioxidants and consider whether it is effective in the aged model.

Mitochondrial Energy Metabolism in the Regulation of Thermogenic Brown Fats and Human Metabolic Diseases.

Brown fats specialize in thermogenesis by increasing the utilization of circulating blood glucose and fatty acids. Emerging evidence suggests that brown adipose tissue (BAT) prevents the incidence of obesity-associated metabolic diseases and several types of cancers in humans. Mitochondrial energy metabolism in brown/beige adipocytes regulates both uncoupling protein 1 (UCP1)-dependent and -independent thermogenesis for cold adaptation and the utilization of excess nutrients and energy. Many studies on the quantification of human BAT indicate that mass and activity are inversely correlated with the body mass index (BMI) and visceral adiposity. Repression is caused by obesity-associated positive and negative factors that control adipocyte browning, de novo adipogenesis, mitochondrial energy metabolism, UCP1 expression and activity, and noradrenergic response. Systemic and local factors whose levels vary between lean and obese conditions include growth factors, inflammatory cytokines, neurotransmitters, and metal ions such as selenium and iron. Modulation of obesity-associated repression in human brown fats is a promising strategy to counteract obesity and related metabolic diseases through the activation of thermogenic capacity. In this review, we highlight recent advances in mitochondrial metabolism, thermogenic regulation of brown fats, and human metabolic diseases.

Transcriptome analysis reveals brown adipogenic reprogramming in chemical compound-induced brown adipocytes converted from human dermal fibroblasts.

Brown adipogenesis contributes to controlling systemic energy balance by enhancing glucose and lipid consumptions. We have previously reported chemical compound-induced brown adipocytes (ciBAs) directly converted from human dermal fibroblasts using a serum-free medium. In this study, genome-wide transcriptional analysis was performed in ciBAs in comparison with the control fibroblasts. A broad range of integrated gene expression was enhanced in functional groups including tricarboxylic acid cycle, electron transfer chain, triglycerides metabolism, fatty acid and glucose metabolism, and adaptive thermogenesis. The results suggested that the chemical conversion underwent metabolic and mitochondrial reprogramming closely associated with functions in brown/beige adipocytes. Moreover, we also compared the transcriptional changes to those of adipocyte browning in adipose tissue-derived mesenchymal stem cells (AdMSCs). Transcriptome analysis indicated that the same sets of metabolic and mitochondria-related genes were similarly changed in the adipocyte browning. Interestingly, ciBAs more expressed Ucp1, while AdMSC-derived adipocytes predominantly expressed Ucp2. UCP1 protein was also more expressed in ciBAs than in AdMSC-derived adipocytes. Based on the evidence that UCP1, but not UCP2, is responsible for adrenergic thermogenesis, ciBAs could be a promising model for human beige adipocytes applicable for basic research, drug development, and clinical uses.

Clinical Benefits of Antioxidative Supplement Twendee X for Mild Cognitive Impairment: A Multicenter, Randomized, Double-Blind, and Placebo-Controlled Prospective Interventional Study.

Oxidative stress is part of the entire pathological process that underlies the development of Alzheimer's disease (AD), including the mild cognitive impairment (MCI) stage. Twendee X (TwX) is a supplement containing a strong antioxidative mix of eight antioxidants, which has been shown to have a clinical and therapeutic benefit in AD model mice. Here, we conducted a multicenter, randomized, double-blind, and placebo-controlled prospective interventional study to evaluate the efficacy of TwX in mitigating MCI. The primary outcomes were differences in Mini-Mental State Examination (MMSE) and Hasegawa Dementia Scale-revised (HDS-R) scores between baseline and six months for placebo and TwX groups. Seventy-eight subjects with MCI were randomized into placebo (n = 37) and TwX (n = 41) groups. MMSE scores at six months differed significantly between the TwX and placebo groups (p = 0.018), and HDS-R scores for the TwX group exhibited a significant improvement at six months relative to baseline (p = 0.025). The TwX group did not show any change in affective or activities of daily living scores at six months. The present study indicates that strong antioxidative supplement TwX is clinical beneficial for cognitive function in subjects with MCI.

Chemical compound-based direct reprogramming for future clinical applications.

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy.

Antioxidant action of fermented grain food supplement: Scavenging of peroxyl radicals and inhibition of plasma lipid oxidation induced by multiple oxidants.

Unregulated oxidative modification of biological molecules induced by multiple oxidants in vivo has been implicated in the pathogenesis of various diseases. Accordingly, the role of antioxidants contained in foods in the maintenance of health and prevention of diseases has received much attention. The efficacy of antioxidants against oxidative stress depends on the nature of oxidants. In the present study, the antioxidant action of fermented grain food supplement, Antioxidant Biofactor (AOB), for scavenging peroxyl radical and inhibition of plasma lipid oxidation induced by multiple oxidants was measured. The antioxidant efficacy against lipid oxidation was assessed by the level of lipid hydroperoxides produced using diphenyl-1-pyrenylphosphine, which is not fluorescent per se but reacts with lipid hydroperoxides stoichiometrically to yield highly fluorescent diphenyl-1-pyrenylphosphine oxide. AOB acted as a potent peroxyl radical scavenger and suppressed lipid oxidation induced by peroxyl radical, peroxynitrite, hypochlorite, and singlet oxygen, but not by 15-lipoxygenase.

Direct conversion of human fibroblasts to brown adipocytes by small chemical compounds.

Brown adipocytes play an important role in human energy metabolism and prevention of obesity and diabetes. Induced pluripotent stem cells (iPSCs) represent a promising source for brown adipocytes; however, exogenous gene induction is generally required for iPSCs generation, which might cause undesired effects particularly in long-term treatment after transplantation. We have previously reported a cocktail of six small chemical compounds that enables a conversion of human fibroblasts into chemical compound-induced neuronal cells (CiNCs). Here, we report that modified combinations of the chemical compounds and rosiglitazone, a PPARγ agonist, afforded direct conversion of human fibroblasts into brown adipocytes. The chemical compound-induced brown adipocytes (ciBAs) exhibit induction of human brown adipocyte-specific genes such as Ucp1, Ckmt1, Cited1 and other adipocyte-specific genes such as Fabp4, AdipoQ, and Pparγ. Treatment with either isoproterenol or Forskolin further induced the expression of Ucp1, suggesting that ß adrenergic receptor signalling in ciBAs could be functional for induction of thermogenic genes. Moreover, oxygen consumption rates were elevated in ciBAs along with increase of cellular mitochondria. Our findings might provide an easily accessible approach for generating human brown adipocytes from fibroblasts and offer therapeutic potential for the management of obesity, diabetes, and related metabolic disorders.

Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1.

The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects.

Partially hydrolysed guar gum ameliorates murine intestinal inflammation in association with modulating luminal microbiota and SCFA.

Partially hydrolysed guar gum (PHGG), a water-soluble dietary fibre produced by the controlled partial enzymatic hydrolysis of guar gum beans, has various physiological roles. This study aimed to elucidate the beneficial effects of PHGG on colonic mucosal damage in a murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Acute colitis was induced in male C57BL/6 mice with TNBS after 2 weeks of pre-feeding with PHGG (5 %). The colonic mucosal inflammation was evaluated using macroscopic damage scores, and neutrophil infiltration was assessed by measuring tissue-associated myeloperoxidase (MPO) activity in the colonic mucosa. TNF-α expression in the colonic mucosa was measured by ELISA and real-time PCR. Moreover, the intestinal microbiota and production of SCFA were assessed by real-time PCR and HPLC, respectively. Colonic damage due to TNBS administration was significantly ameliorated by PHGG treatment. Furthermore, PHGG significantly inhibited increases in MPO activity and TNF-α protein and mRNA expression in the colonic mucosa in TNBS-induced colitis. On analysis of intestinal microbiota, we found that the concentration of the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum subgroup (Clostridium cluster IV) and the Bacteroides fragilis group had significantly increased in PHGG-fed mice. On analysis of SCFA, we found that the caecal content of acetic acid, propionic acid and butyric acid had significantly increased in PHGG-fed mice. Together, these results suggest that chronic ingestion of PHGG prevents the development of TNBS-induced colitis in mice by modulating the intestinal microbiota and SCFA, which may be significant in the development of therapeutics for inflammatory bowel disease.

Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical ß-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial ß-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.

Multiple targets of carbon monoxide gas in the intestinal inflammation.

Inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease are chronic relapsing and remitting inflammatory disorders of the intestinal tract. It is important to investigate the precise pathogenesis of IBD, to evaluate new anti-inflammatory agents, and to develop novel drugs. Carbon monoxide (CO) has emerged as an important regulator of acute and chronic inflammation of the gastrointestinal tract. The mechanism underlying its anti-inflammatory effects is only partially understood. Recent reports have demonstrated that CO could play a role in the functional modulation of epithelial and immunological cells in the intestine. In this short review, we have highlighted the recent findings that CO stimulates the epithelial cell restitution and FGF production from myofibroblasts. CO was also shown to regulate T cell activation and differentiation, and to activate macrophages. Finally, we have discussed the direction of translational research with respect to launching a novel agent for releasing CO in the intestine.

Protective effect of agaro-oligosaccharides on gut dysbiosis and colon tumorigenesis in high-fat diet-fed mice.

High-fat diet (HFD)-induced alteration in the gut microbial composition, known as dysbiosis, is increasingly recognized as a major risk factor for various diseases, including colon cancer. This report describes a comprehensive investigation of the effect of agaro-oligosaccharides (AGO) on HFD-induced gut dysbiosis, including alterations in short-chain fatty acid contents and bile acid metabolism in mice. C57BL/6N mice were fed a control diet or HFD, with or without AGO. Terminal restriction fragment-length polymorphism (T-RFLP) analysis produced their fecal microbiota profiles. Profiles of cecal organic acids and serum bile acids were determined, respectively, using HPLC and liquid chromatography-tandem mass spectrometry systems. T-RFLP analyses showed that an HFD changed the gut microbiota significantly. Changes in the microbiota composition induced by an HFD were characterized by a decrease in the order Lactobacillales and by an increase in the Clostridium subcluster XIVa. These changes of the microbiota community generated by HFD treatment were suppressed by AGO supplementation. As supported by the data of the proportion of Lactobacillales order, the concentration of lactic acid increased in the HFD + AGO group. Data from the serum bile acid profile showed that the level of deoxycholic acid, a carcinogenic secondary bile acid produced by gut bacteria, was increased in HFD-receiving mice. The upregulation tended to be suppressed by AGO supplementation. Finally, results show that AGO supplementation suppressed the azoxymethane-induced generation of aberrant crypt foci in the colon derived from HFD-treated mice. Our results suggest that oral intake of AGO prevents HFD-induced gut dysbiosis, thereby inhibiting colon carcinogenesis.

Identification of cysteinylated transthyretin, a predictive biomarker of treatment response to partially hydrolyzed guar gum in type 2 diabetes rats, by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

Recent evidence has indicated that total fiber intake is inversely related to type 2 diabetes risk. The present study aimed to investigate the effects of chronic administration of partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber, on the occurrence of diabetes and its complications, fatty liver and nephropathy. We also identified predictive serum biomarkers of treatment response to PHGG by mass spectroscopy-based proteomic analysis using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a good model of human non-insulin-dependent diabetes mellitus. In this study, at 5 weeks of age, OLETF rats and control strain Long-Evans Tokushima Otsuka (LETO) rats were fed a control diet or a high-fiber diet (5% PHGG) for 57 weeks. Body weight, food intake, oral glucose tolerance test, plasma insulin levels, and urine glucose and protein levels were regularly measured. Oral glucose tolerance tests (OGTT) and storage of serum in a deep freezer were conducted at the beginning of the experiment and every 4 weeks after overnight fasting during the experiments. PHGG treatment affected neither meal patterns nor the body weight of OLETF and LETO rats. Repeated measure analysis of variance revealed significant differences in fasting plasma glucose and plasma glucose at 2 h after OGTT between control OLETF (OLETF-C) rats and OLETF rats treated with PHGG (OLETF-F). The glucose response determined by the area under the curve of OGTT was significantly greater in OLETF-C rats than that in OLETF-F rats at 25 weeks of age. HOMA-IR, an index of insulin resistance, increased at 25 weeks of age in OLETF-C rats, while this increase was significantly inhibited in OLETF-F rats. At 62 weeks of age, PHGG treatment significantly improved hepatic steatosis as well as renal mesangial matrix accumulation in OLETF rats. To identify the risk marker for diabetes mellitus by SELDI-TOF MS, we collected sera from 21-week-old individuals. Among the 12 specific peaks that were risk marker candidates for diabetes mellitus, the m/z 13,720 peak was identified as that of cysteinylated transthyretin by sequencing of four tryptic peptides using tandem mass spectrometry and peak distribution around the m/z 13,720 peak in the SELDI-TOF spectra. In conclusion, we found that chronic treatment with PHGG improved insulin resistance, delayed the onset of diabetes, and inhibited the development of diabetic complications, as well as identified cysteinylated transthyretin as a predictive biomarker of treatment response to PHGG in OLETF rats.

Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer.

BACKGROUND: NK cells can destroy tumor cells without prior sensitization or immunization. Tumors often lose expression of MHC molecules and/or antigens. However, NK cells can lyse tumor cells in a non-MHC-restricted manner and independent of the expression of tumor-associated antigens. NK cells are therefore considered ideal for adoptive cancer immunotherapy; however the difficulty of obtaining large numbers of fully functional NK cells that are safe to administer deters its clinical use. This phase I clinical trial seeks to address this obstacle by first developing a novel system that expands large numbers of highly activated clinical grade NK cells, and second, determining if these cells are safe in a mono-treatment so they can be combined with other reagents in the next round of clinical trials. METHODS: Patients with unresectable, locally advanced and/or metastatic digestive cancer who did not succeed with standard therapy were enrolled. NK cells were expanded ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296 induced T cells. Patients were administered autologous natural killer cell three times weekly via intravenous infusions in a dose-escalating manner (dose 0.5 × 10(9), 1.0 × 10(9), 2.0 × 10(9) cells/injection, three patients/one cohort). RESULTS: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population. Consequently, NK cells were expanded to approximately 4720-fold (range 1372-14,116) with cells being highly lytic in vitro and strongly expressing functional markers such as NKG2D and CD16. This NK cell therapy was very well tolerated with no severe adverse events. Although no clinical responses were observed, cytotoxicity of peripheral blood was elevated approximately twofolds up to 4 weeks post the last transfer. CONCLUSION: We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells. We also determined that the expanded cells were safe to administer in a monotherapy and are suitable for the next round of clinical trials where their efficacy will be tested combined with other reagents. TRIAL REGISTRATION: UMIN UMIN000007527.

Correlated expression levels of endothelin receptor B and Plexin C1 in melanoma.

Discussion concerning the effect of endothelin receptor B (Ednrb) on melanoma continues because Ednrb has been reported to have both tumor promoting and suppressive effects for melanoma. In order to examine Ednrb-related signaling in melanomagenesis, DNA microarray analysis for a melanoma from a RFP/RET-transgenic mouse (RET-mouse) and a melanoma from an Ednrb-heterozygously deleted RET-mouse [Ednrb(+/-);RET-mouse], in both of which melanoma spontaneously develops, was performed in this study. We found that the expression level of Plexin C1 (PlxnC1), a suppressor for melanoma, in a melanoma from an Ednrb(+/-);RET-mouse was drastically decreased compared to that in a melanoma from a RET-mouse. Therefore, we further examined the correlation between Ednrb and PlxnC1 expression levels in melanomas. PlxnC1 transcript expression levels in melanomas from Ednrb(+/-);RET-mice were lower than those in melanomas from RET-mice. A strong correlation between Ednrb and PlxnC1 transcript expression levels (R = 0.78, p < 0.01) was also found in melanomas from both RET-mice and Ednrb(+/-);RET-mice. Correspondingly, there was a significant correlation between transcript (R = 0.80; p < 0.01) and protein (R = 0.60; p < 0.01) expression levels of EDNRB and PLXNC1 in human primary melanomas. Together with our results showing that the expression level of PLXNC1 transcript was reduced in EDNRB-depleted human melanoma cells, our results showing positively correlated expression levels of Ednrb/EDNRB and PlxnC1/PLXNC1 in melanoma suggest that PlxnC1/PLXNC1 is involved in the Ednrb/EDNRB-mediated suppressive effect on melanoma.

Assessment of radical scavenging capacity of antioxidants contained in foods and beverages in plasma solution.

The assessment of the radical scavenging capacity of antioxidants has been the subject of extensive studies and controversy. The aim of this study is to develop a simple and inexpensive method for the assessment of the radical scavenging capacity of antioxidants contained in foods and beverages in plasma solution, a biologically relevant heterogeneous medium. Three types of probes, hydrophilic pyranine, with low reactivity, hydrophilic pyrogallol red (PGR), with high reactivity, and lipophilic BODIPY, with moderate reactivity, were separately used to measure the amount and rate of peroxyl radical scavenging. The amount of radicals scavenged by antioxidants was assessed from the lag phase produced by antioxidants in the decay of pyranine and BODIPY, while the reactivity of the antioxidants was assessed from their effect on the decay rate of PGR. Two liquid and two solid samples were tested. Commercial bottled green tea and vegetable juice were found to scavenge 15.6 and 3.45 mmol radicals L(-1) and the former scavenged peroxyl radicals 81 times faster than the latter. As for the solid samples, instant coffee powder was found to scavenge several times more radicals and more rapidly than green tea powder. This method may be applied to the assessment of the radical scavenging capacity of antioxidants contained in foods, beverages, and supplements in biologically relevant heterogeneous media.