IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells

We examined whether interleukin (IL)-6 exerts the stimulatory effects on the secretion of histamine from human mast cells triggered by crosslinking of the high affinity IgE receptor (FcepsilonRI) with IgE and anti-IgE. As target cells, we used peripheral blood-derived cultured mast cells grown with SCF, because they were superior in FcepsilonRIalpha expression to cord blood-derived mast cells. Incubation with SCF+IL-6 for 1 week increased the IgE-dependent release as well as intracellular content of histamine in the cultured mast cells, as compared with the values obtained by incubation with SCF alone. The magnitude of these increases was higher than that for priming with SCF+IL-4. A striking difference was also found in the expression of FcepsilonRIalpha between the two-factor combinations. The addition of IL-6 during FcepsilonRI crosslinking with IgE/anti-IgE in the presence of SCF did not influence histamine secretion. When SCF, IL-6 and IL-4 were used together, a further increase was observed in the anti-IgE-dependent liberation of histamine from the cultured mast cells, compared with the two-factor combinations. These results suggest that IL-6 functions as a secretagogue for the inflammatory mediator of human mast cells in the presence of SCF.     

The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring (3)H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC(50) values in the HMC-1.1 subclone lacking KIT D816V (0.025 μM) and the HMC-1.2 subclone expressing KIT D816V (0.005 μM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1(nu) mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC(50) 0.5-1 μM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.     

Mammary hypertrophy is not associated with increased estrogen receptors

Estrogen promotes secondary female sex characteristics, including breast enlargement. Since excessive breast hypertrophy is unrelated to elevated serum estrogen levels, it has been postulated that the enlarged breast is a hypersensitive "target organ." At the cellular level, estrogen crosses the cell membrane, is bound to a cytoplasmic estrogen receptor (ER), and induces the formation of specific anabolic proteins. In breast cancer, this estrogen receptor is regarded as a measure of the sensitivity of the cell to estrogen. To determine if mammary hypertrophy is related to an increase in the number of estrogen receptors, we assayed breast tissue, not fat, from 25 consecutive breast reductions. The median age of patients was 26 years (17 to 77 years), with 752 gm per breast removed on average. Twenty-four percent of the patients were taking estrogens, primarily birth control bills. Cellular estrogen-receptor status was measured by a standardized cytosol extraction radioactive estradiol technique. Estrogen receptors were undetectable (less than 3 fmol/mg cytosol protein) in all patients. We conclude that estrogen receptors alone, and hence estrogen, are not a determinant in mammary hypertrophy. If the enlarged breast is a "target organ," it is by another mechanism.     

DNA methylation is not increased in mouse-human somatic cell hybrids

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Subcutaneous obesity is not associated with sympathetic neural activation

We tested the hypothesis that muscle sympathetic nerve activity (MSNA) would not differ in subcutaneously obese (SUBOB) and nonobese (NO) men with similar levels of abdominal visceral fat despite higher plasma leptin concentrations in the former. We further hypothesized that abdominal visceral fat would be the strongest body composition- or regional fat distribution-related correlate of MSNA among these individuals. To accomplish this, we measured MSNA (via microneurography), body composition (via dual-energy X-ray absorptiometry), and abdominal fat distribution (via computed tomography) in 15 NO (body mass index 0.05, respectively) despite approximately 2.6-fold higher (P < 0.05) plasma leptin concentration in the SUBOB men. Furthermore, abdominal visceral fat was the only body composition- or regional fat distribution-related correlate (r = 0.45; P < 0.05) of MSNA in the pooled sample. In addition, abdominal visceral fat was related to MSNA in NO (r = 0.58; P = 0.0239) but not SUBOB (r = 0.39; P = 0.3027) men. Taken together with our previous observations, our findings suggest that the relation between obesity and MSNA is phenotype dependent. The relation between abdominal visceral fat and MSNA was evident in NO but not in SUBOB men and at levels of abdominal visceral fat below the level typically associated with elevated cardiovascular and metabolic disease risk. Our observations do not support an obvious role for leptin in contributing to sympathetic neural activation in human obesity and, in turn, are inconsistent with the concept of selective leptin resistance.     

Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-Adenosyl-methionine and S-Adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM (10(-5) M) was shown to induce the membrane phospholipid methylation as assessed by the 3H-methyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation.     

Hydroxyl radical-induced apoptosis in human tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as *OH and telomerase/telomere. To address the interrelations between *OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with *OH radicals generated via Fe(2+)-mediated Fenton reactions (0.1 mM FeSO(4) plus 0.3-0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during *OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that *OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of *OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during *OH-induced apoptosis. Strikingly, we also found that *OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that *OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway.     

RNA is closely associated with human mast cell lipid bodies

Both novel and multiple ultrastructural studies based on different principles show relationships of cytoplasmic lipid bodies and ribonucleic acid (RNA) of potential importance to RNA metabolism in human mast cells. The methods include general ultrastructural morphological observations, imaging of RNA with an EDTA regressive stain, imaging of the incorporation of radio labeled uridine by ultrastructural autoradiography, postembedding immunogold labeling of uridine, ribosomes and small nuclear ribonuclear proteins and ultrastructural in situ hybridization detection of poly(A)-positive messenger RNA. Altogether these studies implicate human mast cell lipid bodies in RNA metabolism and are analogous to earlier similar studies which showed that human mast cell granules also curtain RNA.     

Rosiglitazone is not associated with an increased risk of bladder cancer

BackgroundWhether rosiglitazone may increase bladder cancer risk has not been extensively investigated.MethodsThe reimbursement databases of all Taiwanese diabetic patients under oral anti-diabetic agents or insulin from 1996 to 2009 were retrieved from the National Health Insurance. An entry date was set at 1 January 2006 and a total of 885,236 patients with type 2 diabetes were followed up for bladder cancer incidence till end of 2009. Incidences for ever-users, never-users and subgroups of rosiglitazone exposure (using tertile cutoffs of time since starting rosiglitazone, duration of therapy and cumulative dose) were calculated and hazard ratios estimated by Cox regression.ResultsThere were 102,926 ever-users and 782,310 never-users, respective numbers of incident bladder cancer 356 (0.35%) and 2753 (0.35%), and respective incidence 98.3 and 101.6 per 100,000 person-years. The overall hazard ratios (95% confidence intervals) did not show significant association in unadjusted model [0.969 (0.867, 1.082)] and models adjusted for age and sex [0.983 (0.880, 1.098)] or all covariates [0.980 (0.870, 1.104)]. Neither the P values for the hazard ratios for the different categories of the dose–responsive parameters, nor their P-trends were significant.ConclusionsRosiglitazone does not increase the risk of bladder cancer.     

Fetal lung and placental methylation is associated with in utero nicotine exposure

In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10^?03), ANKRD33B (P = 3.12 × 10^?03), CNTD2 (P = 4.9 × 10^?03) and DPP10 (P = 5.43 × 10^?03) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10^?06 ? 3.48 × 10^?05). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.     

Fetal lung and placental methylation is associated with in utero nicotine exposure

In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10⁻⁰³), ANKRD33B (P = 3.12 × 10⁻⁰³), CNTD2 (P = 4.9 × 10⁻⁰³) and DPP10 (P = 5.43 × 10⁻⁰³) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10⁻⁰⁶ − 3.48 × 10⁻⁰⁵). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.     

Active plasma phospholipid transfer protein is associated with apoA-I- but not apoE-containing lipoproteins

Plasma phospholipid transfer protein (PLTP) is a multifaceted protein with diverse biological functions. It has been shown to exist in both active and inactive forms. To determine the nature of lipoproteins associated with active PLTP, plasma samples were adsorbed with anti-A-I, anti-A-II, or anti-E immunoadsorbent, and PLTP activity was measured in the resulting plasma devoid of apolipoprotein A-I (apoA-I), apoA-II, or apoE. Anti-A-I and anti-A-II immunoadsorbents removed 98 +/- 1% (n = 8) and 38 +/- 25% (n = 7) of plasma PLTP activity, respectively. In contrast, only 1 +/- 5% of plasma PLTP activity was removed by anti-E immunoadsorbent (n = 7). Dextran sulfate (DS) cellulose did not bind apoA-I, but it removed 83 +/- 5% (n = 4) of the PLTP activity in plasma. In size-exclusion chromatography, PLTP activity removed by anti-A-I or anti-A-II immunoadsorbent was associated primarily with particles of a size corresponding to HDL, whereas a substantial portion of the PLTP activity dissociated from DS cellulose was found in particles larger or smaller than HDL. These data show the following: 1) active plasma PLTP is associated primarily with apoA-I- but not apoE-containing lipoproteins; 2) active PLTP is present in HDL particles with and without apoA-II, and its distribution between these two HDL subpopulations varies widely among individuals; and 3) DS cellulose can remove active PLTP from apoA-I-containing lipoproteins, and this process creates new active PLTP-containing particles in vitro.     

Prenatal exposure to neurotoxic metals is associated with increased placental glucocorticoid receptor DNA methylation

Epigenetic alterations related to prenatal neurotoxic metals exposure may be key in understanding the origins of cognitive and neurobehavioral problems in children. Placental glucocorticoid receptor (NR3C1) methylation has been linked to neurobehavioral risk in early life, but has not been examined in response to neurotoxic metals exposure despite parallel lines of research showing metals exposure and NR3C1 methylation each contribute to a similar set of neurobehavioral phenotypes. Thus, we conducted a study of prenatal neurotoxic metals exposure and placental NR3C1 methylation in a cohort of healthy term singleton pregnancies from Rhode Island, USA (n = 222). Concentrations of arsenic (As; median 0.02 ug/g), cadmium (Cd; median 0.03 μg/g), lead (Pb; median 0.40 μg/g), manganese (Mn; median 0.56 μg/g), mercury (Hg; median 0.02 μg/g), and zinc (Zn; 145.18 μg/g) measured in infant toenails were categorized as tertiles. Multivariable linear regression models tested the independent associations for each metal with NR3C1 methylation, as well as the cumulative risk of exposure to multiple metals simultaneously. Compared to the lowest exposure tertiles, higher levels of As, Cd, Pb, Mn, and Hg were each associated with increased placental NR3C1 methylation (all P<0.02). Coefficients for these associations corresponded with a 0.71–1.41 percent increase in NR3C1 methylation per tertile increase in metals concentrations. For Zn, the lowest exposure tertile compared with the highest tertile was associated with 1.26 percent increase in NR3C1 methylation (P=0.01). Higher cumulative metal risk scores were marginally associated with greater NR3C1 methylation. Taken together, these results indicate that prenatal exposure to neurotoxic metals may affect the offspring's NR3C1 activity, which may help explain cognitive and neurodevelopmental risk later in life.     

Heat-shock protein 72 cell-surface expression on human lung carcinoma cells is associated with an increased sensitivity to lysis mediated by adherent natural killer cells

 The cell-surface expression patterns of major histocompatibility complex (MHC) class I, class II and heat-shock protein 72 (HSP72) molecules were measured on human lung (LX-1) and mammary (MX-1) carcinoma cells. No major differences were found in the MHC cell-surface expression pattern of both cell lines. However, they differ significantly in their capacity to express HSP72 on their cell surface. Under physiological conditions LX-1 cells express HSP72 molecules on more than 90% of the cells, whereas MX-1 cells exhibit no significant HSP72 cell-surface expression (less than 5%). These expression patterns remained stable in all further cell passages tested. The sensitivity to lysis mediated by an interleukin-2 (IL-2)-stimulated, adherent natural killer (NK) cell population could be correlated with the amount of cell-surface-expressed HSP72 molecules. By antibody-blocking studies, using HSP72-specific monoclonal antibody (mAb), a strong inhibition of lysis was only found with LX-1 cells but not with MX-1 cells. In contrast to the cell-surface expression, the cytoplasmic amount of HSP72 in MX-1 cells was twice as high compared to LX-1 cells under physiological conditions. After nonlethal heat-shock the rate of induction and the total cytoplasmic amounts of HSP72 were comparable in both cell lines. The clonogenic cell viability of LX-1 cells after incubation at temperatures ranging from 41°C to 44°C was significantly elevated compared to that of MX-1 cells. In conclusion we state the following: (i) HSP72 cell-surface expression on human carcinoma cells is independent of the cytoplasmic amount of HSP72; (ii) the cell-surface expression of HSP72 is associated with an increased sensitivity of tumour cells to lysis mediated by an IL-2-stimulated, adherent NK cell population; (iii) thermoresistance is not related to the cytoplasmic HSP72 level but might be related to the amount of HSP72 expressed on the cell surface. Received: 20 June 1996 / Accepted: 25 September 1996