Usefulness of Ninjin'yoeito for Chronic Obstructive Pulmonary Disease Patients with Frailty.

Objectives: Frail patients with chronic obstructive pulmonary disease (COPD) have a higher risk of mortality, mood disorder, and poor quality of life (QOL). There are few intervention studies in frail patients with COPD, and there is a need for an effective therapy. Ninjin'yoeito (NYT) is a Kampo medicine that has been reported to improve fatigue, psychosomatic vulnerability, and respiratory symptoms. We examined the efficacy of NYT in frailty or prefrailty patients with COPD. Design: Prospective, single-center, open-label, randomized controlled trial. Location: Showa University Hospital, Tokyo, Japan. Subjects: Sixty-two patients (53 males and 9 females) with a mean age of 76 ± 6 years were included in the analysis. Interventions: The patients were divided into two groups: the NYT group (n = 31) and the control (standard treatment) group (n = 31). Outcome measures: The primary outcome was changes in Kihon checklist (KCL) scores at week 24, which reflect changes in frailty. The secondary outcomes were changes in the following assessment scores at week 24: Simplified Nutritional Appetite Questionnaire (SNAQ) scores, which reflect changes in appetite; COPD Assessment Test (CAT) scores, which reflect changes in QOL in patients with COPD; Hospital Anxiety and Depression Scale (HADS)-Anxiety scores, which reflect changes in anxiety; and HADS-Depression scores, which reflect changes in depression. Results: There was a slight but not significant difference in changes in KCL scores between the NYT and control groups (p = 0.09). However, there were statistically significant differences in changes in SNAQ (p = 0.03), CAT (p = 0.03), HADS-Anxiety (p < 0.01), and HADS-Depression (p = 0.02) scores between the two groups. Conclusions: Our results suggest that NYT is an effective and promising drug with various effects in patients with COPD who are frail, despite conventional treatment.

Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells.

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α­fetoprotein (AFP) and albumin were examined by reverse transcription­quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all­trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all­trans retinoic acid and insulin­transferrin­selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post­stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.

An Optimal Medium Supplementation Regimen for Initiation of Hepatocyte Differentiation in Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem (hiPS) cells are an ideal source for hepatocytes. Glucose and arginine are necessary for cells to survive. Hepatocytes have galactokinase (GALK), which metabolizes galactose for gluconeogenesis, and ornithine transcarbamylase (OTC), which converts ornithine to arginine in the urea cycle. Hepatocyte selection medium (HSM) lacks both glucose and arginine, but contains galactose and ornithine. Although human primary hepatocytes survive in HSM, all the hiPS cells die in 3 days. The aim of this study was to modify HSM so as to initiate hepatocyte differentiation in hiPS cells within 2 days. Hepatocyte differentiation initiating medium (HDI) was prepared by adding oncostatin M (10 ng/ml), hepatocyte functional proliferation inducer (10 nM), 2,2'-methylenebis (1,3-cyclohexanedione) (M50054) (100 µg/ml), 1× non-essential amino acid, 1× sodium pyruvate, nicotinamide (1.2 mg/ml), L-proline (30 ng/ml), and L-glutamine (0.3 mg/ml) to HSM. HiPS cells (201B7 cells) were cultured in HDI for 2 days. RNA was isolated, used as template for cDNA, and subjected to real-time quantitative polymerase chain reaction. Alpha-fetoprotein, γ-glutamyl transpeptidase, and delta-like 1 were upregulated. Expression of albumin was not observed. Expression of transcription factors specific to hepatocytes was upregulated. The expression of GALK2, OTC, and CYP3A4 were increased. In conclusion, differentiation of 201B7 cells to hepatoblast-like cells was initiated in HDI. Limitations were small number of cells were obtained, and the cells with HDI were not mature hepatocytes.

Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells.

Induced pluripotent stem (iPS) cells are ideal sources of hepatocyte for transplantation into patients experiencing hepatic failure. Growth and transcription factors were analyzed to design a single-step protocol for the differentiation of iPS cells into hepatocytes. The expression of transcription factors was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and compared among iPS cells, as well as fetal and adult liver cells. iPS cells were cultured with growth factors and RT-PCR was performed to analyze the expression of transcription factors. iPS cells were introduced with transcription factors, cultured with growth factors and subjected to real-time quantitative PCR. Indocyanine green (ICG) was added to the medium as a hepatocyte marker. Sox17, GATA4, GATA6, FoxA2, HEX, HNF4α and C/EBPα were expressed in fetal and adult liver cells, but not in iPS cells. Sox17, GATA6 and HNF4α were expressed after exposure a combination of oncostatin M, epidermal growth factor, retinoic acid, dexamethasone and ITS (OERDITS). When iPS cells were introduced with FoxA2, GATA4, HEX and C/EBPα and cultured with OERDITS for 8 days, the cells expressed α-fetoprotein, δ-like (Dlk)-1 and γ-glutamyl transpeptidase (GTP), and ICG uptake was observed. Exposure to FoxA2, GATA4, HEX and C/EBPα and culturing with OERDITS supplementation potentially serves as a single-step inducer for the differentiation of iPS cells into hepatic progenitor-like cells within 8 days.

Effects of arginine treatment on nutrition, growth and urea cycle function in seven Japanese boys with late-onset ornithine transcarbamylase deficiency.

BACKGROUND: The aim of this study was to investigate the effects of arginine on nutrition, growth and urea cycle function in boys with late-onset ornithine transcarbamylase deficiency (OTCD). Seven Japanese boys with late-onset OTCD enrolled in this study resumed arginine treatment after the cessation of this therapy for a few years. Clinical presentations such as vomiting and unconsciousness, plasma amino acids and urinary orotate excretion were followed chronologically to evaluate urea cycle function and protein synthesis with and without this therapy. In addition to height and body weight, blood levels of proteins, lipids, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein -3 (IGFBP-3) were monitored. RESULTS: The frequency of hyperammonemic attacks and urinary orotate excretion decreased significantly following the resumption of arginine treatment. Despite showing no marked change in body weight, height increased gradually. Extremely low plasma arginine increased to normal levels, while plasma glutamine and alanine levels decreased considerably. Except for a slight increase in high-density lipoprotein cholesterol level, blood levels of markers for nutrition did not change. In contrast, low serum IGF-I and IGFBP-3 levels increased to age-matched control levels, and normal urinary GH secretion became greater than the level observed in the controls. CONCLUSION: Arginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.