Mechanism of palmitic acid-induced deterioration of in vitro development of porcine oocytes and granulosa cells.

The concentration of fatty acids in follicular fluid reflect the physical condition of donors, and palmitic acid (PA) is a major component of follicular fluid. The present study examined the effect of PA on in vitro oocyte growth and investigated the molecular backgrounds of the PA induced-low quality oocytes. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of gilts. The OGCs were cultured for 14 days in a medium containing 0.5 mM PA or vehicle (BSA). PA was found to reduce granulosa cell (GCs) proliferation (0.73 fold) and viability (93.9% vs. 85.8%) and increase lipid content in oocytes and GCs. Oocytes developed in the presence of PA had low developmental ability to the blastocyst stage. In addition, PA affected developmental and epigenetic markers of histone modifications in oocytes; levels of H4K12 acetylation and H3K9 demethylation. PA affected cellular proliferation, apoptosis and endoplasmic reticulum stress markers along with reducing the phosphor-AKT/AKT levels and increasing the expression levels of caspase-3 and CHOP in GCs. Incubation of OGCs with PA increased ceramide content in the GC, and addition of ceramide to the culture medium inhibited GC proliferation. In conclusion, it is suggested that high PA content in the medium reduces viability and proliferation through ceramide accumulation, and PA impaires the developmental ability of oocytes grown in vitro. In addition, high-fat conditions induce changes in the histone modifications of oocytes grown in vitro.

Randomised phase II trial of irinotecan plus S-1 in patients with gemcitabine-refractory pancreatic cancer.

BACKGROUND: We aimed to compare the efficacy and safety of irinotecan/S-1 (IRIS) therapy with S-1 monotherapy in patients with gemcitabine-refractory pancreatic cancer. METHODS: Patients were treated with oral S-1 (80-120 mg for 14 days every 4 weeks) plus intravenous irinotecan (100 mg m-2 on days 1 and 15 every 4 weeks; IRIS group) or oral S-1 group (80-120 mg daily for 28 days every 6 weeks). The primary endpoint was progression-free survival (PFS). RESULTS: Of 137 patients enrolled, 127 were eligible for efficacy. The median PFS in the IRIS group and S-1 monotherapy group were 3.5 and 1.9 months, respectively (hazard ratio (HR)=0.77; 95% confidence interval (CI), 0.53-1.11; P=0.18), while the median overall survival (OS) were 6.8 and 5.8 months, respectively (HR=0.75; 95% CI, 0.51-1.09; P=0.13). Response rate was significantly higher in the IRIS group than in the S-1 monotherapy group (18.3% vs 6.0%, P=0.03). Grade 3 or higher neutropenia and anorexia occurred more frequently in the IRIS group. CONCLUSIONS: There was a trend for better PFS and OS in the IRIS group that could be a treatment arm in the clinical trials for gemcitabine-refractory pancreatic cancer.

Tsunami inundation after the Great East Japan Earthquake and mortality of affected communities.

OBJECTIVE: To examine the relationship between mortality rate and tsunami inundation after the Great East Japan Earthquake (GEJE) in 2011. STUDY DESIGN: Cross-sectional study. METHODS: One hundred and fifty-five town or village sections in Ishinomaki, Myagi Prefecture, were included in this study. Three areas in the city were classified by characteristic landforms: plains area (n = 114), ria coastal area (n = 27) and Kitakami riverside (n = 14). The correlation coefficient between tsunami inundation depth and mortality rate was calculated for each area, and the differences between the areas were examined. Furthermore, multivariate analyses adjusted for the characteristics of the sections were conducted using census data taken before the GEJE. RESULTS: An association was found between inundation depth and mortality rate for Ishinomaki as a whole (r = 0.65, P < 0.001), Kitakami riverside (r = 0.85, P < 0.001) and the plains area (r = 0.75, P < 0.001) in separate analyses. However, no association was detected between inundation depth and mortality rate for the ria coastal area (r = 0.14, P = 0.47). CONCLUSION: The ria coastal area has good accessibility to the hills and tight bonding between members of the community. These factors seemed to play crucial roles in the lower mortality rate in this area despite the deep inundation.

Randomised phase II study comparing dose-escalated weekly paclitaxel vs standard-dose weekly paclitaxel for patients with previously treated advanced gastric cancer.

BACKGROUND: This randomised phase II trial compared dose-escalated weekly paclitaxel (wPTX) vs standard-dose wPTX for patients with previously treated advanced gastric cancer (AGC). METHODS: Ninety patients were randomised to a standard dose of wPTX (80 mg m(-2)) or an escalated dose of wPTX (80-120 mg m(-2)) to assess the superiority of overall survival (OS) with a one-sided alpha error of 0.3 and a power of 0.8. RESULTS: The median OS showed a trend towards longer survival in the dose-escalated arm (11.8 vs 9.6 months; hazard ratio (HR), 0.75; one-sided P=0.12), although it was statistically not significant. The median progression-free survival (PFS) was significantly longer in the dose-escalated arm (4.3 vs 2.5 months, HR, 0.55; P=0.017). Objective response rate was 30.3% with dose escalation and 17.1% with standard dose (P=0.2). The frequency of all grades of neutropenia was significantly higher with dose escalation (88.7% vs 60.0%, P=0.002); however, no significant difference was observed in the proportion of patients experiencing grade 3 or more (40.9% vs 31.1%, P=0.34). CONCLUSION: Dose-escalated wPTX in patients with pretreated AGC met our predefined threshold of primary end point, OS (P<0.3); however, it did not show a significantly longer OS. Progression-free survival was significantly better with dose escalation.

Development of serum IgM antibodies against superantigens of Staphylococcus aureus and Streptococcus pyogenes in Kawasaki disease.

To serologically determine the association of microbial superantigens and the pathogenesis of Kawasaki disease (KD), we conducted a case-control study. Serum IgG and IgM antibodies against staphylococcal enterotoxin A (SEA), SEB, SEC, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxin A (SPEA) were measured by an enzyme-linked immunosorbent assay in 293 serum samples from 65 KD patients on clinical days 1-28 and 120 control samples. The administration of immunoglobulin products, which contain high concentrations of IgG antibodies against all the superantigens, directly elevated antitoxin IgG antibodies in KD patients. In contrast, antitoxin IgM antibodies were not detected in immunoglobulin products. Actually, we found a significant elevation of IgM antibodies against SEA in KD patients in the first (median titre: 0.020, P < 0.01 versus control), second (0.024, P < 0.001), third (0.030, P < 0.001) and fourth (0.038, P < 0.001) weeks, compared to the controls (0.015). Significant differences of IgM antibodies were also true for SEB, TSST-1, and SPEA throughout the first to fourth weeks, and for SEC throughout the second to fourth weeks. The prevalence of KD patients having high IgM titres (> mean + 2SD of control values) to the 5 superantigens was increased with the clinical weeks, and reached 29-43% of KD subjects at the fourth week. This is the first study that describes kinetics of IgM antibodies against superantigens and clarifies the serological significance throughout the clinical course of KD. Our results suggest that multiple superantigens involve in the pathogenesis of KD.

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome.

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.

Transient elevation of interleukin-16 levels at the initial stage of meningitis in children.

IL-16 is an immunomodulatory cytokine that is characterized by chemotactic activity and stimulation of proinflammatory cytokine expression in monocytic cells. We studied IL-16 using ELISA in children with meningitis. When meningeal symptoms existed, IL-16 levels were high in the cerebrospinal fluid (CSF) of both bacterial (939 +/- 877 ng/l, n = 20) and aseptic (341 +/- 371 ng/l, n = 23) meningitis. The values in the CSF were significantly higher than those in non-meningitis controls (29 +/- 8 ng/l, n = 22, P < 0.0001). After meningeal symptoms disappeared, IL-16 levels in bacterial (191 +/- 149 ng/l, n = 10, P = 0.0042) and aseptic (159 +/- 188 ng/l, n = 13, P = 0.0118) meningitis were lower than those during the symptomatic stage. IL-16 levels were the highest before day 5 of the illness and then gradually fell. Significant correlations were found between IL-16 levels and both G-CSF levels (r = 0.783, n = 11, p = 0.0029) and IL-6 levels (r = 0.818, n = 12, P = 0.0005) in the CSF of bacterial and aseptic meningitis. IL-16 levels in all CSF samples from non-meningitis controls were lower than those in serum. In contrast, IL-16 levels in the CSF in six of 16 samples from bacterial meningitis and two of 18 samples from aseptic meningitis were higher than those in serum. Serum levels of IL-16 did not fluctuate throughout the course of meningitis. These data indicate that IL-16 levels rise transiently in CSF at the initial stage of meningitis. We speculate that IL-16 may promote inflammatory responses during meningitis in concert with other proinflammatory cytokines.

Antigenic components of Malassezia species for immunoglobulin E antibodies in sera of patients with atopic dermatitis.

Antigenic components of Malassezia furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis (AD). Antigenic components were extracted from Malassezia cells by treatment with beta-mercaptoethanol, referred to as 2-ME extract. CBB staining and lectin blots using Con A, LCA, PHA-E4, PNA or RCA120 showed that the 2-ME extracts contained several species-dependent components that differed quantitatively and qualitatively among the Malassezia species at the protein level. In the Western blot with the 2-ME extracts, of 54 sera of the patients with AD (54 patients), the patients' IgE antibodies most frequently recognized components showing molecular weights of 43-46 kDa for M. slooffiae, 12-22 kDa for M. sympodialis, 35-40 kDa for M. restricta, 45-50 kDa for M. globosa, and of 67-72 kDa for M. furfur, respectively. In the correlative study, in which the total band intensities generated for each extract in Western blot were compared among the Malassezia species, the intensity for M. globosa was well correlated with that for M. sympodialis (r=0.756). In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patient's IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level.

[Association of idiopathic thrombocytopenic purpura and type 1 diabetes mellitus in a patient with sarcoidosis].

Occurrence of autoimmune diseases with sarcoidosis is well known. However, a case in which more than one of these diseases coexist with sarcoidosis is rare. We present a young man with suspected sarcoidosis, complicated by idiopathic thrombocytopenic purpura (ITP) and type 1 A diabetes mellitus (DM). A 21-year-old man was admitted to our hospital because of thrombocytopenia, hyperglycemia, and bilateral hilar lymphadenopathy (BHL). Although a histological proof could not be obtained, the patient was considered to have sarcoidosis because 67-gallium scintigraphy disclosed "Lambda" and "Panda" signs which are highly specific for sarcoidosis. Type 1 A DM was also diagnosed as the patient had antiglutamic acid decarboxylase antibodies. The patient disclosed no hepatosplenomegaly or no lymphadenopathy and diagnosis of ITP was confirmed by bone marrow examination. High dose steroid was started as the thrombocytopenia progressed. The platelet number increased satisfactorily and shrinkage of BHL was also observed with the therapy.

An increase in circulating mast cell colony-forming cells in asthma.

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

Antioxidant activities of buckwheat hull extract toward various oxidative stress in vitro and in vivo.

We have undertaken four basic in vitro studies and an animal experiment to obtain information about the antioxidant activities of buckwheat hull extract (BWHE). In the in vitro studies, BWHE scavenged super oxide anion produced in the xanthine/xanthine oxidase system (IC50=11.4 microg phenolic compound/ml), and strongly inhibited autoxidation of linoleic acid (IC50=6.2 microg phenolic compound/ml). Low-density lipoprotein (LDL) oxidation induced by Cu2+ ion was also protected by BWHE. In the animal experiment, ddY mice were fed a standard diet supplemented with 0.75% BWHE for 14 d. In blood, liver and brain of the mice TBARS and fluorescent substance concentration were significantly decreased compared with those of non-treated mice. SOD like activity in serum also significantly rose by BWHE treatment. BWHE was shown to be effective for protecting biological systems against various oxidative stresses in vitro, and to have antioxidant activity in vivo.

Early elevation of serum thrombopoietin levels and subsequent thrombocytosis in healthy preterm infants.

To verify pathophysiological mechanisms underlying thrombocytosis in low-birth-weight (LBW) preterm babies, we evaluated kinetic changes in platelet counts and thrombopoietic cytokines including thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 in 24 uncomplicated preterm infants. Platelet counts in cord blood (CB) (265 +/- 64 x 10(9)/l) were similar to adult levels, increased by d 14 (473 +/- 140 x 10(9)/l), and then remained fairly constant. Thrombocytosis (> 500 x 10(9)/l) was observed in 9/24 (38%) subjects. Mean TPO level in CB was 5.11 +/- 1.51 fmol/ml, peaked at d 2 (7.64 +/- 3.28 fmol/ml), decreased at d 5 (3.93 +/- 1.67 fmol/ml), and thereafter kept fairly constant during the remaining neonatal period. Compared with term infants, mean TPO levels of preterm infants in CB and at d 2 were significantly higher (P < 0.01). There was an inverse correlation between platelet counts and TPO levels (r = 0.45, P < 0.001, n = 88). Preterm neonates with thrombocytosis had significantly higher TPO values in CB than those without thrombocytosis (P < 0.05). There was no significant relationship between platelet counts and IL-6. IL-11 was not detectable. These results suggest that an early elevation of serum TPO levels is related to the subsequent thrombocytosis in LBW preterm infants.

H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate.

A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.

Stressful delivery influences circulating thrombopoietin (TPO) levels in newborns: possible role for cortisol in TPO-mpl binding.

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl(+)/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system.

Isolation and characterization of a major antigenic component of Malassezia globosa to IgE antibodies in sera of patients with atopic dermatitis.

Three major components of Malassezia globosa were isolated from 2-ME extracts of this fungus by ion-exchange column chromatography and are referred to as Malg46a, Malg46b and Malg67, respectively. IgE antibodies to these components in the sera of patients with AD were detected by immunoblots. In Western blot, IgE antibodies to Malg46b were most frequently detected in the sera of AD patients. Dot blot with the Malg46b-containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of the patients who were positive for IgE antibodies to the 2-ME extract of M. globosa in the Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in the Western blot (r=0.763). In the lectin blot, Con A reacted with both Malg46a and Malg46b but not with Malg67. The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. In conclusion, a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.