Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs.

Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases.

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD) and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-ß; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.

Selective down-regulation of Th2 cell-mediated airway inflammation in mice by pharmacological intervention of CCR4.

BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.

New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus.

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.

Effect of fermented liquid diet prepared with Lactobacillus plantarum LQ80 on the immune response in weaning pigs.

Probiotics such as lactic acid bacteria directly influence the host's health and have beneficial effects such as decreasing the number of enteric pathogens, regulating intestinal immune responses and preventing diseases. Among domestic animals, probiotics have been expected to be an alternative to antibiotics added in the diet; and fermented liquid diet (FLD) containing probiotics has great potential as a diet for reducing the use of antibiotics. In this study, we evaluated the immunomodulatory effects of FLD, prepared using Lactobacillus plantarum LQ80 (LQ80), on the immune response of weaning pigs. Ten weaning piglets were divided into two groups and were fed the FLD (n = 5) or a non-fermented liquid diet (NFLD) (n = 5) for 28 days. At the end of the experiment, the total immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the sera of the FLD-fed piglets were significantly higher than those of the NFLD-fed piglets (P < 0.05). In contrast, the total immunoglobulin A (IgA) levels in the feces and saliva were not significantly affected by FLD feeding. However, the mean fecal IgA levels of FLD-fed piglets at day 28 were higher than those at 14 and 21 days (P < 0.05). Blood cells from the FLD-fed piglets showed a low level of interferon-γ secretion and mitogen-induced proliferation compared to that of the NFLD-fed piglets. Furthermore, the levels of interluekin-8 and tumor necrosis factor-α, which are proinflammatory cytokines, in the blood cells of the FLD-fed piglets were lower than those of the NFLD-fed piglets (P < 0.05). In conclusion, the FLD used in this study could alter the immune responses of weaning piglets by stimulation of the systemic or mucosal antibody response, without unnecessary inflammatory reactions. This indicates, that the FLD feed prepared with the use of LQ80 may be a candidate feed, with regard to enhancing immune responses and preventing diseases in weaning piglets.

Haplotype structures of the UGT1A gene complex in a Japanese population.

Genetic polymorphisms of UDP-glucuronosyltransferases (UGTs) are involved in individual and ethnic differences in drug metabolism. To reveal co-occurrence of the UGT1A polymorphisms, we first analyzed haplotype structures of the entire UGT1A gene complex using the polymorphisms from 196 Japanese subjects. Based on strong linkage disequilibrium between UGT1A8 and 1A10, among 1A9, 1A7, and 1A6, and between 1A3 and 1A1, the complex was divided into five blocks, Block 8/10, Block 9/6, Block 4, Block 3/1, and Block C, and the haplotypes for each block were subsequently determined/inferred. Second, using pyrosequencing or direct sequencing, additional 105 subjects were genotyped for 41 functionally tagged polymorphisms. The data from 301 subjects confirmed the robustness of block partitioning, but several linkages among the haplotypes with functional changes were found across the blocks. Thus, important haplotypes and their linkages were identified among the UGT1A gene blocks (and segments), which should be considered in pharmacogenetic studies.

Metabolism and urinary excretion of a new quinoline anticancer drug, TAS-103, in humans.

1. TAS-103, a novel condensed quinoline derivative, has been developed as an anticancer drug targeting topoisomerases I and II. 2. The purpose of the present study was to characterize the metabolism and urinary excretion of TAS-103 after the intravenous infusion of a single dose to patients in Phase I clinical trials. 3. Five metabolites were detected using high-performance liquid chromatography (HPLC) photodiode array and a precursor scan by liquid chromatography mass spectrometry mass spectrometry (LC/MS/MS). 4. Structures of the five metabolites were determined using the results of enzymatic hydrolysis and the analysis of production mass spectra obtained by LC/MS/MS, and by comparing HPLC retention times and UV, mass and production mass spectra of authentic standards. 5. The metabolites were identified as demethyl-TAS-103 glucuronide (DM-TAS-103-G), TAS-103 glucuronide (TAS-103-G), TAS-103 glucuronide N-oxide (NO-TAS-103-G), demethyl-TAS-103 (DM-TAS-103) and TAS-103 N-oxide (NO-TAS-103). 6. The mean total amount of TAS-103 and TAS-103-G in urine was only 6.03% of the dose, suggesting that urine is not the main elimination route. TAS-103 was extensively metabolized, and a small percentage of the parent drug (0.41%) was found in urine.

Apparent low frequency of sequence variability within the proximal promoter region of the cytochrome P450 (CYP) 3A5 gene in established cell lines from Japanese individuals.

The members of the cytochrome P450 (CYP) 3A subfamily play an important role in the metabolism of more than 50% of the drugs metabolized by CYPs. Among the CYP3A members, CYP3A5 is known to exhibit polymorphic expression within the human liver. We hypothesized that a single nucleotide polymorphism (SNP) in the 5'-regulatory region of the CYP3A5 gene might be the cause of CYP3A5 polymorphic expression. Due to the existence of "CYP3AP1," a highly homologous sequence to the CYP3A5 gene, it was necessary to make specific primers to the CYP3A5 gene. In the present study, we designed a series of oligonucleotide primers for sequencing the proximal promoter region of the CYP3A5 gene in order to search for the putative regulatory single nucleotide polymorphism. We examined 86 established cell lines derived from Japanese individuals as a representation of the Japanese population. However, no SNP was detected in the promoter region of the CYP3A5 gene isolated from the cell lines used, suggesting other causal factors for the observed polymorphism of CYP3A5-dependent drug metabolism.

Effects of HSP-117, a novel tachykinin NK1-receptor antagonist, on cisplatin-induced pica as a new evaluation of delayed emesis in rats.

The effects of a novel tachykinin NK1-receptor antagonist HSP-117 [(2S,3S)-3-[(5-isopropyl-2,3-dihydrobenzofuran-7-yl)methyl]amino-2-phenylpiperidine dihydrochloride] on cisplatin-induced pica, i.e., the eating of nonnutritive substances such as kaolin were examined in rats. HSP-117 inhibited kaolin intake in a dose-dependent manner for 2 days. The 5-HT3-receptor antagonist ondansetron inhibited only on the first day, but not on the second day. These results indicate that the cisplatin-induced kaolin intake on the first day is related to both 5-HT3- and NK1 receptors, while only the NK1 receptor is involved on the second day. Thus, cisplatin-induced continuous pica in rats represents a useful model of not only acute but also delayed emesis.

[Estimation of daily dietary intake of aluminum].

The daily dietary intake of aluminum was estimated through a total diet study from 1996 to 1998. In ten institutes, total diet study samples were prepared and their aluminum concentration was determined. The average daily intake of aluminum was 3.5 mg and the range was 1.8-8.4 mg. The validity of the analytical result was supported by analyses of certified reference materials.

[Assessment of intra and extrahepatic perfusion during hepatic artery infusion chemotherapy using slow-injected gadolinium-enhanced MR imaging through implanted catheter-port-system].

Twenty-four studies of intra-arterially slow-injected gadolinium-enhanced MR imaging through an implanted catheter-port system (reservoir-MR) were carried out in 15 patients with liver tumor. The flow rate of gadolinium injection was 0.1 ml/sec and a total of 3 mL was injected. Six consecutive phases, each with an acquisition time of 14 seconds, were obtained every 30 seconds. In all studies, the signal intensity of the drug delivery portion became very high. Twenty-three of 24 studies showed intrahepatic perfusion in the first phase. The hepatic vein was enhanced at the first phase in 10 and the second phase in 14. The abdominal aorta was enhanced at the second phase in all 24 studies. The portal vein was enhanced at the first phase in 4, the second phase in 13, and the third phase in 7 studies. Both intra- and extrahepatic perfusion were more clearly demonstrated by reservoir-MRI than by digital subtraction angiography through an implanted catheter-port system (reservoir-DSA); however, morphological changes in the hepatic artery were better demonstrated by reservoir-DSA than by reservoir-MRI.

[Ultra-slow infusion dynamic MRI using an infusion pump: a new method for the evaluation of drug distribution in arterial infusion chemotherapy].

Ultra slow infusion dynamic MR imaging using an infusion pump(IP-MRI) was performed in six patients with metastatic liver tumor or unresectable gastric cancer to evaluate ant-cancer drug distribution. We used un implanted port for the infusion of Gd-DTPA by infusion pump. On IP-MRI, the speed of Gd-DTPA infusion was very slow (0.01 ml/sec) , the same as drug infusion at chemotherapy. The contrast enhancement of tumors was extremely clear. Therefore, IP-MRI was considered feasible for the evaluation of drug distribution.

Inhibition by nifedipine of adherence- and activated macrophage-induced death of human gingival fibroblasts.

The effects of nifedipine on the death and proliferation of gingival fibroblasts were investigated to elucidate the mechanism of gingival overgrowth that is associated with chronic administration of Ca2+ channel blockers. The number of adhered viable and dead fibroblasts obtained from healthy human gingiva increased after confluence, whereas cell death was inhibited by nifedipine in a concentration-dependent manner. A similar inhibition was also observed in the presence of other calcium channel blockers, such as nicardipine, diltiazem, and verapamil. When gingival fibroblasts were co-cultured with RAW264 (macrophage-like) cells, lipopolysaccharide (LPS) caused the concentration-dependent death of fibroblasts. Nifedipine significantly inhibited the LPS-induced cell death. Although neither LPS nor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitroso-hydrazino)-ethanamine, a nitric oxide donor, directly caused fibroblast death, 3-morpholino-sydnonimine (SIN-1), a peroxynitrite donor, induced fibroblast death, regardless of the presence of RAW cells. The cell death induced by SIN-1 was not affected by nifedipine treatment. LPS stimulation caused an increase in the immunoreactivity of inducible nitric oxide synthase (iNOS) and in the nitrite concentration in the incubation medium of RAW cells. The induction of iNOS was completely prevented by the incubation with nifedipine. The inhibition by nifedipine of nitrite production in RAW cells was also observed after treatment with nicardipine, but not with either diltiazem or verapamil. Therefore, the inhibition by nifedipine of both adherence- and LPS-stimulated macrophage-induced death of fibroblasts may be the mechanism of gingival overgrowth seen during chronic treatment with Ca(2+) channel blockers.

Decreases in phenytoin hydroxylation activities catalyzed by liver microsomal cytochrome P450 enzymes in phenytoin-treated rats.

Phenytoin, 5,5-diphenylhydantoin, is a widely used anticonvulsant agent with a variety of toxicities, including drug interactions. The formation of four oxidative metabolites, 4'-hydroxylated (4'-HPPH), 3'-hydroxylated (3'-HPPH), a catechol (3',4'-diHPPH), and the 3',4'-dihydrodiol form of phenytoin was examined in rat liver microsomes. In 11 cDNA-expressed rat P450 enzymes tested, CYP2C6 had the highest activities in 4'- and 3'-HPPH formation from phenytoin, followed only by CYP2C11. In contrast, CYP2C11 had high activity for 3',4'-diHPPH formation from 4'-HPPH, followed by CYP2C6. The rates of 4'-HPPH and 3',4'-diHPPH formation from phenytoin in liver microsomes in the presence of NADPH were significantly decreased by oral administration of phenytoin (300 mg/kg for 20 days) to rats, despite the increase in P450 contents. However, the cumene hydroperoxide-supported formation of 3',4'-dihydrodiol and 4'-HPPH from phenytoin was induced by phenytoin administration. Hydrogen peroxide formation in reaction mixtures with NADPH was induced by the administration of phenytoin; however, the coupling ratio of phenytoin oxidation was decreased in phenytoin-induced liver microsomal P450 systems. These results suggested that phenytoin could not stimulate its own apparent oxidative metabolism by liver P450s induced with phenytoin administration. The increase of unmetabolized phenytoin and byproducts of oxygen generated in the phenytoin-induced liver microsomal P450 system may be involved in phenytoin-related drug toxicity.

Plasma concentrations of interleukin-1beta, interleukin-6, soluble interleukin-2 receptor and tumor necrosis factor alpha of depressed patients in Japan.

There are a number of investigations which indicate the important relationship between depression and cytokines. In this study, we investigated plasma interleukin (IL)-1beta, IL-6, soluble IL-2 receptor (sIL-2R) and tumor necrosis factor (TNF)-alpha of depressed patients whose clinical evaluation was performed by the Hamilton Rating Scale for Depression (HAM-D) and the Profile of Mood States (POMS). They were compared with those of the control subjects, and before and after treatment with antidepressants. Before the treatment, plasma IL-1beta, IL-6, sIL-2R and TNF-alpha of the patients were not significantly different from those of the control subjects. sIL-2R was positively correlated with the POMS-tension-anxiety subscale and tended to have a positive correlation with HAM-D. After pharmacotherapy, TNF-alpha levels of the depressed patients increased, without any relationship between the change in the HAM-D or the POMS and the change in TNF-alpha. These results suggest that the plasma sIL-2R concentration is associated with mood state, and that the plasma TNF-alpha concentration is increased after pharmacotherapy in Japanese depressed patients.

Problems during and after pregnancy of former biliary atresia patients treated successfully by the Kasai procedure.

BACKGROUND/PURPOSE: The aim of this study was to investigate the problems and the quality of life during and after pregnancy of the patients who had undergone Kasai operation and to find out a strategy for follow-up during the period of their pregnancy. METHODS: A questionnaire was sent to 134 institutions of the Japanese Biliary Atresia Society with the following questions: (1) Do you have any pregnancy cases in patients who had undergone Kasai operation? (2) Did she have any menstrual problem? (3) Did she have any problem during pregnancy and delivery? (4) Did she have any change in liver function tests after delivery? (5) Did she have any early and long-term problem after delivery? (6) Did the baby have any problem? (7) Was there any special care or comment about the pregnancy of the biliary atresia patients? The responses were analyzed. RESULTS: Fourteen institutions reported 16 cases of pregnancy, 23 cases of delivery, and 2 cases of abortion. The causes of abortion in the 2 cases were attributed to hemorrhagic shock after massive bleeding from esophageal varices and serious atopic dermatitis, respectively. Other problems during pregnancy were abruption of placenta, fetal distress leading to caesarian section, and development of liver dysfunction leading liver transplantation. Problems after delivery included deterioration of liver function in 6 patients (37.5%), attacks of ascending cholangitis in 4 patients (25.0%), and severe fatigue with liver dysfunction from nursing the baby leading to liver transplantation. Only 3 of 16 (18.8%) patients were free of any problems. No abnormality was seen in the babies. CONCLUSIONS: Even if the patients with biliary atresia lead a good postoperative course, unexpected complications can occur when they become pregnant. Close long-term follow-up is required for proper management of pregnancy in biliary atresia patients.

Rupture of a giant saccular aneurysm of coronary arteriovenous fistulas.

A 58-year-old Japanese woman was admitted to our hospital because of chest pain. A continuous murmur was detected at the left parasternal area. Electrocardiogram showed ST elevation in leads V2, V3 and V4. Chest computed tomography and echocardiography demonstrated pericardial effusion and a large mass which was adjacent to the pulmonary artery. An abnormal blood flow was detected in the mass by Doppler echocardiography. Coronary angiography confirmed that the mass was a giant aneurysm of coronary arteriovenous fistula arising from both the left and right coronary arteries. This patient had no symptoms until rupture of the fistula. Rupture of a coronary arteriovenous fistula is very rare but can be a cause of chest pain and pericardial effusion.

Surgical treatment of neuroblastoma with micrometastasis.

BACKGROUND/PURPOSE: The aim of this study was to define the role of surgery in neuroblastoma with micrometastasis, which is detectable only by the polymerase chain reaction (PCR) method. METHODS: Fifty samples (peripheral blood 9, bone marrow 41) were harvested sequentially from 27 neuroblastoma patients, and the micrometastases were examined using the previously described single-step PCR method. The results were reviewed with the clinical courses. RESULTS: Radical surgery was performed in 9 patients with bone marrow micrometastasis. Event-free survival was obtained in 2 patients with stage IV disease (25.0%) for a follow-up period of 2 to 6 years in this group. Both patients received intraoperative radiation and subsequent autologous bone marrow transplantation (ABMT) using purged marrow. Radical surgery was performed in 18 patients without micrometastasis, and 6 of 9 advanced patients (66.7%) survived without the disease including 4 patients who received unpurged stem cell transplantation. CONCLUSIONS: Persistent micrometastasis in bone marrow should be considered predictive as a poor prognostic factor in neuroblastoma. Intensive local control with surgery and radiation is important for the patients with micrometastasis and should be followed by ABMT using purged marrow. Unpurged marrow may be safely used if the single-step PCR detects no micrometastasis.

Psychometric properties of the Japanese version of the Mental Adjustment to Cancer (MAC) scale.

This paper describes a study of the psychometric properties of the Japanese version of the mental adjustment to cancer (MAC) scale developed in England. The scale was completed by 455 Japanese cancer patients. The internal consistency was similar to that of the original version (Cronbach's alpha coefficients for the subscales ranged from 0.60 to 0.78) and the Japanese version had moderate to moderately high stability (correlation coefficients were above 0.64). Correlations between the MAC scale score and emotional states measured by the Profile of Mood States (POMS) indicated the concurrent validity. The results suggest that the Japanese version, like the original MAC scale, is a reliable and valid clinical research tool in Japan.