Association between dexamethasone treatment and alterations in serum concentrations of trace metals.

Previously, a significant elevation in the serum levels of iron (Fe) was observed within a few days after the initiation of cisplatin (CDDP)-based chemotherapy. To clarify the underlying mechanisms, the serum concentration of hepcidin, a negative regulator of Fe release, was determined in the clinical samples obtained from six patients with cancer. The result showed that the serum concentration of hepcidin in patients receiving CDDP-based chemotherapy was significantly increased after 4-6 days of treatment, in comparison to the baseline level, suggesting that aforementioned excessive systemic Fe was not explained by the change of serum hepcidin level. All these patients received antiemetic premedication. We next evaluated of the effects of Pt-containing drugs and prophylactic antiemetic dexamethasone medication on the serum concentration of trace metals in mice, and on the hepatic and renal concentration of trace metals. The serum concentrations of Fe, Cu, and Zn in the CDDP-treated and oxaliplatin-treated mice were not significantly altered in comparison to those of the vehicle-treated control group. The serum concentrations of Fe, Cu, and Zn were increased after 24 h of dexamethasone treatment, compared to those of the control group (P < 0.05). The hepatic concentration of Mn was significantly reduced, whereas those of Fe and Cu inclined to diminish. The present findings suggest that dexamethasone can partly contribute to the changes in the serum concentrations of trace metals during anticancer chemotherapy.

A new high-throughput analysis for drug metabolism profiling using liquid chromatography coupled with tandem mass spectrometry.

We developed 3 liquid chromatography (LC) methods coupled to tandem mass spectrometry for comprehensive high-throughput profiling of drug metabolism, employing different columns with gradient systems of aqueous 10 mmol/L ammonium acetate and acetonitrile. The methods were established using testosterone (TST), imipramine (IMP), acetaminophen (APAP) and salbutamol (SBM), which have markedly different values of partition coefficient, P. Method I, using an octadecylized silica (ODS) column (ACQUITY UPLC BEH C18) with a steep gradient, was suitable for analyzing TST and its metabolites, and IMP and its metabolites. These substrates could not be analyzed by Method II, using another ODS column (Atlantis dC18), or Method III, using an Atlantis HILIC silica column. APAP and its metabolites could be analyzed simultaneously by Method II, but not Method I or III. SBM and its metabolites could be detected simultaneously only by Method III. The developed analytical methods were further evaluated with a panel of 18 additional drugs. Based on the retention times and peak shapes, we propose general criteria, in terms of calculated LogP (cLogP) value, for method selection. Broadly speaking, drugs with high cLogP, those with intermediate cLogP and those with low cLogP were best analyzed by Methods I, II and III, respectively, although drugs with borderline values of cLogP could be evaluated by both the relevant methods. The injection cycle for each method was within 10 min, including up to 3 min conditioning time. These methods are expected be useful in high-throughput screening assays to examine biotransformations of candidate drugs.

Association of genetic variants for susceptibility to obesity with type 2 diabetes in Japanese individuals.

AIMS/HYPOTHESIS: In populations of East Asian descent, we performed a replication study of loci previously identified in populations of European descent as being associated with obesity measures such as BMI and type 2 diabetes. METHODS: We genotyped 14 single nucleotide polymorphisms (SNPs) from 13 candidate loci that had previously been identified by genome-wide association meta-analyses for obesity measures in Europeans. Genotyping was done in 18,264 participants from two general Japanese populations. For SNPs showing an obesity association in Japanese individuals, we further examined diabetes associations in up to 6,781 cases and 7,307 controls from a subset of the original, as well as from additional populations. RESULTS: Significant obesity associations (p < 0.1 two-tailed, concordant direction with previous reports) were replicated for 11 SNPs from the following ten loci in Japanese participants: SEC16B, TMEM18, GNPDA2, BDNF, MTCH2, BCDIN3D-FAIM2, SH2B1-ATP2A1, FTO, MC4R and KCTD15. The strongest effect was observed at TMEM18 rs4854344 (p = 7.1 × 10(-7) for BMI). Among the 11 SNPs showing significant obesity association, six were also associated with diabetes (OR 1.05-1.17; p = 0.04-2.4 × 10(-7)) after adjustment for BMI in the Japanese. When meta-analysed with data from the previous reports, the BMI-adjusted diabetes association was found to be highly significant for the FTO locus in East Asians (OR 1.13; 95% CI 1.09-1.18; p = 7.8 × 10(-10)) with substantial inter-ethnic heterogeneity (p = 0.003). CONCLUSIONS/INTERPRETATION: We confirmed that ten candidate loci are associated with obesity measures in the general Japanese populations. Six (of ten) loci exert diabetogenic effects in the Japanese, although relatively modest in size, and independently of increased adiposity.

Transfection of human HGF plasmid DNA improves limb salvage in Buerger's disease patients with critical limb ischemia.

AIM: Hepatocyte growth factor is a potent angiogenic agent. This study investigated the efficacy and safety of intramuscular injection of naked plasmid DNA encoding the human hepatocyte growth factor gene in Japanese patients with Buerger's disease and critical limb ischemia. METHODS: An open-label clinical study was performed at eight hospitals in Japan from May 2004 to April 2008. Ten patients were enrolled. They had Buerger's disease with ischemic ulcers, were not candidates for revascularization, and were unresponsive to conventional drug therapy. Treatment consisted of 8 injections (total dose: 4 mg) of hepatocyte growth factor plasmid, which were administered into the calf muscles and/or distal thigh muscles of the ischemic limbs under ultrasound guidance. Administration was done twice at an interval of 4 weeks. If there was no improvement after 2 doses, a 3rd dose could be administered. The response to treatment was evaluated from the reduction of ischemic ulcer size. RESULTS: The size of ischemic ulcers showed a decrease in 6/9 (66.7%) patients and the ulcers healed completely in 5/9 (55.6%) patients after gene therapy. Major amputation was not required. There were no deaths and no major safety concerns. CONCLUSION: Hepatocyte growth factor gene therapy is safe and effective for critical limb ischemia in patients with Buerger's disease.

Evaluation of the antiobesity effects of an amino acid mixture and conjugated linoleic acid on exercising healthy overweight humans: a randomized, double-blind, placebo-controlled trial.

This single-centre, randomized, double-blind, placebo-controlled trial investigated the effects of administering a mixture of four amino acids (lysine, proline, alanine and arginine) with or without conjugated linoleic acid to healthy overweight humans before and after exercising. Forty-one healthy subjects (body mass index >or= 23 to < 30 kg/m(2)) completed the study following randomization to receive either placebo or one of three test supplements: amino acid mixture 0.76 g/day; amino acid mixture 1.52 g/day; or amino acid mixture 1.52 g/day coadministered with conjugated linoleic acid 1.6 g/day. Each of the study treatments was administered 30 min before and immediately after a period of daily exercise, which was delivered by an exercise expert, for a period of 12 weeks. When compared with the placebo group, several indicators, such as waist and hip circumferences, were found to have significantly decreased in the test supplement groups compared with the placebo. These results suggest that ingestion of these supplements might enhance the fat-burning effects of exercise.

Randomized, double-blind, placebo-controlled clinical trial of hepatocyte growth factor plasmid for critical limb ischemia.

Hepatocyte growth factor (HGF) is a potent angiogenic factor. The efficacy and safety of intramuscular injection of a naked plasmid encoding human HGF gene (beperminogene perplasmid, Collategene) was investigated in patients with critical limb ischemia (CLI) in a multicenter, randomized, double-blind, placebo-controlled trial. The randomization ratio for plasmid to placebo was 2:1. Injection sites were selected in each patient limb based on angiographic findings. Placebo or plasmid was injected on days 0 and 28. Evaluation of efficacy was carried out after 12 weeks. The primary end point was the improvement of rest pain in patients without ulcers (Rutherford 4) or the reduction of ulcer size in patients with ulcer(s) (Rutherford 5). Secondary end points were ankle-brachial pressure index, amputation, and quality of life (QOL). Forty-four patients were treated, and we performed interim analysis of efficacy in 40 patients. The overall improvement rate of the primary end point was 70.4% (19/27) in HGF group and 30.8% (4/13) in placebo group, showing a significant difference (P=0.014). In Rutherford 5 patients, HGF achieved a significantly higher improvement rate (100% [11/11]) than placebo (40% [2/5]; P=0.018). HGF plasmid also improved QOL. There were no major safety problems. HGF gene therapy is safe and effective for CLI.

Common variants at the GCK, GCKR, G6PC2-ABCB11 and MTNR1B loci are associated with fasting glucose in two Asian populations.

AIMS/HYPOTHESIS: To test fasting glucose association at four loci recently identified or verified by genome-wide association (GWA) studies of European populations, we performed a replication study in two Asian populations. METHODS: We genotyped five common variants previously reported in Europeans: rs1799884 (GCK), rs780094 (GCKR), rs560887 (G6PC2-ABCB11) and both rs1387153 and rs10830963 (MTNR1B) in the general Japanese (n = 4,813) and Sri Lankan (n = 2,319) populations. To identify novel variants, we further examined genetic associations near each locus by using GWA scan data on 776 non-diabetic Japanese samples. RESULTS: Fasting glucose association was replicated for the five single nucleotide polymorphisms (SNPs) at p < 0.05 (one-tailed test) in South Asians (Sri Lankan) as well as in East Asians (Japanese). In fine-mapping by GWA scan data, we identified in the G6PC2-ABCB11 region a novel SNP, rs3755157, with significant association in Japanese (p = 2.6 x 10(-8)) and Sri Lankan (p = 0.001) populations. The strength of association was more prominent at rs3755157 than that of the original SNP rs560887, with allelic heterogeneity detected between the SNPs. On analysing the cumulative effect of associated SNPs, we found the per-allele gradients (beta = 0.055 and 0.069 mmol/l in Japanese and Sri Lankans, respectively) to be almost equivalent to those reported in Europeans. CONCLUSIONS/INTERPRETATION: Fasting glucose association at four tested loci was proven to be replicable across ethnic groups. Despite this overall consistency, ethnic diversity in the pattern and strength of linkage disequilibrium certainly exists and can help to appreciably reduce potential causal variants after GWA studies.

Impact of whole body irradiation and vascular endothelial growth factor-A on increased beta cell mass after bone marrow transplantation in a mouse model of diabetes induced by streptozotocin.

AIMS/HYPOTHESIS: Recent studies have shown that bone marrow transplantation reduces hyperglycaemia in a mouse model of diabetes induced by streptozotocin. However, the essential factors for the improvement of hyperglycaemia by bone marrow transplantation have not been fully elucidated. The aim of this study was to search for such factors. METHODS: We investigated the effect of irradiation to whole body, to abdomen alone or to whole body excluding abdomen, followed by infusion or no infusion of bone marrow cells. We also investigated the effect of bone marrow transplantation on beta cell-specific vascular endothelial growth factor-A gene (Vegfa) knockout mice. RESULTS: Bone marrow transplantation improved streptozotocin-induced hyperglycaemia and partially restored islet mass. This change was associated with increased islet vascularisation. Among the other methods investigated, low-dose irradiation of the whole body without infusion of bone marrow cells also improved blood glucose level. In streptozotocin-treated beta cell-specific Vegfa knockout mice, which exhibit impaired islet vascularisation, bone marrow transplantation neither improved hyperglycaemia, relative beta cell mass nor islet vascularisation. CONCLUSION/INTERPRETATION: Our results indicate that whole body irradiation is essential and sufficient for restoration of beta cell mass after streptozotocin treatment independent of infusion of bone marrow cells. Vascular endothelial growth factor-A produced in beta cells is also essential for this phenomenon.

Association of gastric fluid microbes at birth with severe bronchopulmonary dysplasia.

OBJECTIVE: Gastric fluid microbes were examined in preterm infants at birth to assess their influence on the postnatal outcome. STUDY DESIGN: Prospective cohort study. SETTING: Level III neonatal intensive care unit. PATIENTS: A total of 103 premature neonates with a gestational age of less than 32 weeks. MAIN OUTCOME MEASURE: Gastric fluid microbes were identified by analysis of bacterial 16S ribosomal RNA gene. Additionally, the urease gene of Ureaplasma species was detected by polymerase chain reaction of gastric fluid obtained at birth and/or tracheal aspirate from ventilated preterm infants. The association between detection of microbes and bronchopulmonary dysplasia was investigated through assessment from clinical features and by a lung injury marker (KL-6). RESULTS: Forty-two of 103 gastric fluid specimens were positive for microbes. Ureaplasma species were detected in 23 of the 42 (55%) gastric fluid specimens. All infants with Ureaplasma species in tracheal aspirate fluid also had positive gastric fluid specimens. Compared to infants negative for gastric fluid microbes, infants positive for microbes had higher rates of maternal chorioamnionitis (18% vs 78%), premature rupture of membranes (11% vs 55%), severe bronchopulmonary dysplasia (1.6% vs 14%) and showed higher plasma KL-6 levels during the initial 4 weeks of life. CONCLUSION: Detection of gastric fluid microbes was correlated well with antenatal infection and severe bronchopulmonary dysplasia. Detection of Ureaplasma species in gastric fluid was associated with subsequent respiratory colonisation. These results suggest that antenatal exposure of the immature fetus to microbes may cause lung injury and promote the onset of bronchopulmonary dysplasia.

Dose-dependent requirement of patched homologue 1 in mouse pancreatic beta cell mass.

AIMS/HYPOTHESIS: Ectopic activation of hedgehog (HH) signalling in pancreas induces various abnormal morphogenetic events in the pancreas. This study analysed the dose-dependent requirement of patched homologue 1 (PTCH1), a negative regulator of HH signalling on pancreatic development. METHODS: We used a recessive spontaneous mutant mouse denoted as mes which carries a mutated Ptch1 resulting in deletion of the most carboxy-terminal cytoplasmic domain of the PTCH1 protein. In this study, we analysed pancreatic morphology in Ptch1 ( +/+ ), Ptch1 ( +/mes ), Ptch1 (+/-), Ptch1 ( mes/me ) (s) and Ptch1 (-/mes ) mouse embryos, as well as the islet mass in adult Ptch1 (+/+), Ptch1 (+/mes ) and Ptch1 (+/-) mice. RESULTS: Until embryonic day (E) 12.5, no obvious abnormality of pancreas was observed in any of the Ptch1 mutants. The levels of PDX1 and glucagon were also not evidently different among the mice genotypes studied. Thereafter, morphological abnormalities appeared in the Ptch1 mutant mice. The beta, alpha and exocrine cell masses decreased at E18.5 in parallel with increased HH signalling, with beta cell mass showing the highest sensitivity to HH signalling with a significant decrease even in Ptch1 (+/mes ) mice. Adult Ptch1 (+/-) mice also showed a significant decrease in beta cell mass compared with wild-type mice. CONCLUSIONS/INTERPRETATION: Our findings indicate that the carboxy-terminal domain of Ptch1 is essential for pancreatic development. In addition, the loss of Ptch1 function decreases both the endocrine and exocrine cell mass in a dose-dependent manner, with beta cells particularly sensitive to changes in HH signalling.

Alleviation of Abeta-induced cognitive impairment by ultrasound-mediated gene transfer of HGF in a mouse model.

A new therapeutic approach to treat Alzheimer's disease (AD) is needed, and the use of growth factors is considered to be a candidate. Hepatocyte growth factor (HGF) is a unique multifunctional growth factor, which has the potential effect to exert neurotrophic action and induce angiogenesis. In this study, we examined the effects of overexpression of human HGF plasmid DNA using ultrasound-mediated gene transfer into the brain in an Abeta-infused cognitive dysfunction mouse model. We demonstrated that HGF gene transfer significantly alleviated Abeta-induced cognitive impairment in mice in behavioral tests. These beneficial effects of HGF might be due to (1) significant recovery of the vessel density in the dentate gyrus of the hippocampus, (2) upregulation of BDNF, (3) a significant decrease in oxidative stress and (4) synaptic enhancement. A pharmacological approach including gene therapy to increase the HGF level in combination with anti-Abeta therapy might be a new therapeutic option for the treatment of AD.