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There are at least two phases of β-cell death during the development of autoimmune diabetes: an initiation event that results in the release of β-cell-specific antigens, and a second, antigen-driven event in which β-cell death is mediated by the actions of T lymphocytes. In this report, the mechanisms by which the macrophage-derived cytokine interleukin (IL)-1 induces β-cell death are examined. IL-1, known to inhibit glucose-induced insulin secretion by stimulating inducible nitric oxide synthase expression and increased production of nitric oxide by β-cells, also induces β-cell death.
Methods and Findings
To ascertain the mechanisms of cell death, the effects of IL-1 and known activators of apoptosis on β-cell viability were examined. While IL-1 stimulates β-cell DNA damage, this cytokine fails to activate caspase-3 or to induce phosphatidylserine (PS) externalization; however, apoptosis inducers activate caspase-3 and the externalization of PS on β-cells. In contrast, IL-1 stimulates the release of the immunological adjuvant high mobility group box 1 protein (HMGB1; a biochemical maker of necrosis) in a nitric oxide-dependent manner, while apoptosis inducers fail to stimulate HMGB1 release. The release of HMGB1 by β-cells treated with IL-1 is not sensitive to caspase-3 inhibition, while inhibition of this caspase attenuates β-cell death in response to known inducers of apoptosis.
These findings indicate that IL-1 induces β-cell necrosis and support the hypothesis that macrophage-derived cytokines may participate in the initial stages of diabetes development by inducing β-cell death by a mechanism that promotes antigen release (necrosis) and islet inflammation (HMGB1 release).
Citation: Steer SA, Scarim AL, Chambers KT, Corbett JA (2006) Interleukin-1 Stimulates β-Cell Necrosis and Release of the Immunological Adjuvant HMGB1. PLoS Med 3(2): e17. doi:10.1371/journal.pmed.0030017
Academic Editor: Joel Habener, Harvard Medical School, United States of America.
Received: May 3, 2005; Accepted: October 10, 2005; Published: December 20, 2005
Copyright: © 2006 Steer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AG, aminoguanidine; APC, antigen-presenting cell; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DEANO, sodium (Z)-1(N,N-diethylamino) diazen-1-ium-1,2-diolate; HMGB1, high-mobility group box 1 protein; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NMMA, NG-monomethyl-L-arginine; PS, phosphatidylserine; SEM, standard error of the mean; TNF, tumor necrosis factor; TUNEL, terminal nick-end labeling
Insulin-dependent diabetes is an autoimmune disease characterized by selective destruction of insulin-producing β-cells found in pancreatic islets of Langerhans [1]. The development of diabetes is characterized initially by insulitis, in which leukocytes migrate to and invade islets. This is followed by overt diabetes, distinguished by insulin insufficiency due to the destruction of a majority of β-cells [2,3]. Cytokines produced by inflammatory leukocytes and resident and inflammatory macrophages are believed to contribute to the loss of β-cell function and viability during the development of autoimmune diabetes [4,5]. Interleukin (IL)-1 was identified as the active component of conditioned medium derived from activated human mononuclear cells that causes an inhibition of insulin secretion and the loss of islet cell viability, provided the initial evidence supporting a role for cytokines in the pathogenesis of autoimmune diabetes [6,7].
The mechanisms by which cytokines impair β-cell function have been described in detail. Alone or in combination with interferon (IFN)-γ and tumor necrosis factor (TNF), IL-1 has been shown to stimulate β-cell expression of inducible nitric oxide synthase (iNOS), and the resulting production of nitric oxide impairs β-cell oxidation of glucose to CO2, reduces the activity of the Krebs cycle enzyme aconitase, and reduces islet ATP levels by more than 4-fold [8,9]. Importantly, glucose-induced insulin secretion is dependent on β-cell depolarization and Ca2+ entry, events that are mediated by the inhibition of ATP-sensitive K+ channels due to the accumulation of ATP that is produced by mitochondrial oxidation of glucose to CO2 [10,11]. The targeted disruption of mitochondrial oxidation resulting in the reduction of cellular levels of ATP is one mechanism by which nitric oxide impairs glucose-stimulated insulin secretion. The damaging actions of cytokines on β-cell function appear to be selective for β-cells, as IL-1 fails to impair the ability of α-cells to oxidize glucose to CO2 [12]. Furthermore, β-cells are the primary islet-cell source of iNOS in response to cytokine treatment [13,14]. The macrophage-derived cytokine IL-1 is the major regulator of iNOS expression by β-cells, IL-1 alone is sufficient to stimulate iNOS expression by rat β-cells, and combinations of IL-1 and IFN-γ stimulate β-cell expression of iNOS in most mouse strains and in human islets [4,9,15]. Biochemical and genetic evidence supports a primary role for nitric oxide as the major mediator of cytokine-induced β-cell damage. The NOS inhibitors NG-monomethyl-L-arginine (NMMA) and aminoguanidine (AG) prevent the inhibitory actions of cytokines on insulin secretion, glucose oxidation, and aconitase activity by rat, mouse, and human islets [4,8,9]. In addition, islets isolated from iNOS-deficient mice are resistant to the inhibitory actions of cytokines on insulin secretion [16], and expression of iNOS under control of the rat insulin promoter results in the development of diabetes in a nitric oxide-dependent manner that occurs in the absence of insulitis [17].
While autoimmune diabetes is characterized by selective destruction of β-cells, the biochemical mechanisms by which β-cells are killed are poorly defined. In vitro studies using isolated islets and primary β-cells have shown that nitric oxide is a primary mediator of impaired β-cell function in response to cytokine treatment; however, the role of this free radical as a mediator of β-cell death and the mechanism of cell death has been debated. Much of this debate has centered on whether cytokine-induced islet cell death is apoptotic or necrotic and if nitric oxide is a mediator [18–20]. Early studies by Kolb and coworkers demonstrated that nitric oxide is responsible for IL-1-stimulated DNA damage in β-cells [21]. More recently, cytokines have been reported to induce β-cell apoptosis by a process that requires prolonged exposures of 7–9 d and that appears to occur by nitric oxide-independent mechanisms [19,22]. Similar to the inhibitory actions of cytokines on β-cell function, the macrophage-derived cytokine IL-1 appears to be the primary regulator of cytokine-induced β-cell death, be it by necrosis or apoptosis [18,19,23].
In classical terms, apoptosis is defined as programmed cell death by a pathway that removes unwanted cells in the absence of an inflammatory response. It is a highly organized, energy-dependent process that is characterized by caspase activation, the ordered cellular degradation of proteins and organelles, and maintenance of plasma membrane integrity. The final result is noninflammatory phagocytosis of the dying cell, which minimizes leakage of cellular contents and inflammation [24,25]. In contrast, necrosis is an inflammatory process that is characterized by the loss of membrane integrity and the leakage of cellular contents into the extracellular space. It is an unregulated form of cell death that does not require energy or caspase activation [26]. When considering the role of macrophage-derived cytokines such as IL-1 in the context of the initiation of autoimmune diabetes, one would anticipate that this cytokine would kill β-cells by a mechanism that would promote inflammation. In this report, we compare the effects of IL-1 with known inducers of apoptosis on β-cell death.
Materials and Animals
Sprague Dawley rats were purchased from Harlan (Indianapolis, Indiana, United States). RINm5F (rat insulinoma) cells were obtained from Washington University Tissue Culture Support Center (St. Louis, Missouri, United States). RPMI 1640 containing L-glutamine, CMRL-1066 tissue culture medium, L-glutamine, penicillin, and streptomycin were obtained from GIBCO-BRL (Grand Island, New York, United States). FCS was obtained from Hyclone Laboratories (Logan, Utah, United States). Human recombinant IL-1β was obtained from Cistron Biotechnology (Pine Brook, New Jersey, United States). Sodium (Z)-1(N,N-diethylamino) diazen-1-ium-1,2-diolate (DEANO) was obtained from Alexis (San Diego, California, United States). Enhanced chemiluminescence reagent was purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey, United States). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), neutral red, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), camptothecin, and staurosporine were obtained from Sigma (St. Louis, Missouri, United States). In Situ Cell Death Detection kit was obtained from Roche (Indianapolis, Indiana, United States). Caspase-3 Fluorometric Assay was obtained from R&D Systems (Minneapolis, Minnesota, United States). Annexin V-FITC Apoptosis Detection kit and the caspase-3 inhibitor Z-DQMD-CHO were obtained from Calbiochem (San Diego, California, United States). Guinea pig anti-human insulin antibody was obtained from Linco Research (St. Louis, Missouri, United States). CY3-conjugated donkey anti-guinea pig antibody and HRP-conjugated donkey anti-rabbit and donkey anti-mouse antisera were obtained from Jackson Immunoresearch Laboratories (West Grove, Pennsylvania, United States). Rabbit anti-mouse HMGB1 antibody was obtained from BD Biosciences Pharmingen (San Diego, California, United States). All other reagents were from commercially available sources.
Islet Isolation and Cell Culture
Islets were isolated from 250- to 300-g male Sprague Dawley rats by collagenase digestion as previously described [27]. Islets were cultured overnight in complete CMRL-1066 (containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5.5 mM glucose) at 37 °C under an atmosphere of 95% air and 5% CO2 prior to experimentation. For annexin V and terminal nick-end labeling (TUNEL) assays of cell death, rat islets were treated with IL-1 for the indicated times followed by dispersion into individual cells by trypsin digestion as previously described [27]. RINm5F cells were grown in RPMI 1640 tissue culture medium to a density of ∼75% prior to experimentation. The cells were then removed from growth flasks by treatment with 0.05% trypsin and 0.02% EDTA for 5 min at 37 °C, washed two times with complete CMRL-1066 media, and plated in CMRL-1066 at the indicated density.
Human islets were obtained from the Islet Cell Resource Center at Washington University School of Medicine (St. Louis, Missouri, United States). Before experiments, the human islets were cultured for 48 h at 37 °C in complete CMRL-1066 tissue culture medium in an atmosphere of 95% air and 5% CO2.
Cell Death Assays
Cell viability was examined using the MTT assay and neutral red uptake. The MTT assay is based on the ability of viable cells to reduce MTT to insoluble formazan crystals [28]. For this assay, RINm5F cells or rat islet cells (1 × 105 cells in 200 μl of complete CMRL-1066) were washed with tissue culture medium following treatments, and MTT was added at a final concentration of 0.5 mg/ml. Following an incubation for 3 h at 37 °C, the supernatants were removed and discarded, and the cells were lysed with acid-isopropanol (100 μl of 0.04 N HCl in isopropanol) and mixed thoroughly to dissolve the crystals for 5 min at room temperature. The optical density of this product was determined at a wavelength of 570 nm.
Cell viability was also evaluated by examining the accumulation of neutral red dye in viable cells [29]. For this assay, RINm5F cells or dispersed rat islet cells (1 × 105 cells in 200 μl of complete CMRL-1066) were cultured for 24 or 48 h with the indicated concentrations of IL-1, camptothecin, or staurosporine; the culture supernatants were removed and discarded; and the cells were incubated in fresh medium containing 50 μg/ml neutral red at 37 °C. Following a 2-h incubation, the supernatant was removed, the cells were washed with a 1% formaldehyde-1% CaCl2 solution, and the neutral red dye was extracted in 100 μl of a 50% ethanol-1% acetic acid lysing solution. The accumulation of neutral red dye in the lysing solution was measured at a wavelength of 570 nm. Cell viability using both the MTT and neutral red assays is expressed as the percent of dead cells.
Annexin V-FITC Staining
RINm5F cells or rat islet cells (1 × 105 cells in 200 μl of complete CMRL-1066) were treated as indicated in the figure legends. Cells were removed from plates by cell scraping (RINm5F) or washing (islet cells). Externalization of phosphatidylserine (PS) was evaluated by annexin V staining according to manufacturer's instructions (Calbiochem) and quantified by flow cytometry [30].
TUNEL Staining/Immunohistochemistry
RINm5F cells or rat islet cells (4 × 105 cells in 400 μl of complete CMRL-1066) were treated as indicated in the figure legends. The cells were removed from plates and centrifuged onto glass slides. DNA damage was quantified by TUNEL staining according to the manufacturer's instructions using the In Situ Cell Death Detection Kit, Fluorescein (Roche). Cells containing DNA damage were colocalized with DAPI by incubating cells with DAPI (5 μg/ml) for 15 min followed by three washes with PBS (pH 7.4). DNA damage in rat islet cells was colocalized with insulin-containing cells as outlined previously [31].
Caspase-3 Activity
RINm5F cells or rat islet cells (4 × 105 cells in 400 μl of complete CMRL-1066) were treated as indicated in the figure legends. Cells were isolated and lysed, and capase-3 activity in the cell lysate was determined according to the manufacturer's instructions (Caspase-3 Fluorometric Assay Kit, R&D Systems). Caspase-3 activity was normalized to protein content and is presented as the fold-increase over the level of activity measured in the untreated control.
Western Blot Analysis
Protein lysates prepared from RINm5F cells and rat islets were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Pharmacia Biotech) under semidry transfer conditions as previously described [32]. Antigen was detected by enhanced chemiluminescence according to the manufacturer's specifications (Amersham Pharmacia Biotech). Antibody dilutions were: rabbit anti-mouse HMGB1, 1:1,000; and HRP-conjugated donkey anti-rabbit, 1:7,000.
Nitrite Determination
Nitrite production was determined by mixing 50 μl of cell culture medium with 50 μl of Griess reagent [33]. The absorbance at 540 nm was measured, and nitrite concentrations were calculated from a sodium nitrite standard curve.
Nitric Oxide Mediates Cytokine-Induced Islet Cell Death
While it is clear that the inhibitory actions of cytokines such as IL-1 on glucose-stimulated insulin secretion are mediated by β-cell production of nitric oxide, the role of nitric oxide in islet cell death has been debated [8,18,19]. Short exposures (24–48 h) of islets and insulinoma cell lines to IL-1 alone, or in combination with IFN-γ, and TNF cause islet cell DNA damage and reduced oxidative capacity in a nitric oxide-dependent fashion [34–36]; however, prolonged exposures (7–9 d) of islets or purified β-cells with IL-1 or cytokine combinations have been reported to induce β-cell apoptosis in a nitric oxide-independent manner [19,22]. To investigate the mechanisms of cytokine-induced β-cell death, we initially examined whether IL-1 reduces β-cell viability using the MTT assay. Following a 24-h incubation with IL-1, death of RINm5F cells increased ∼20% (Figure 1A), and this cell death correlated with a ∼10-fold increase in production of nitrite, an oxidative metabolite of nitric oxide (Figure 1B). Following a 48-h incubation with IL-1, RINm5F cell death increased to ∼40% (Figure 1A), and cell death correlated with a ∼20-fold increase in nitrite production (Figure 1B). The NOS inhibitor NMMA prevented nitrite production (Figure 1B) and significantly attenuated IL-1-induced RINm5F cell death (Figure 1A). Similar to its effect in RINm5F cells, IL-1 induced the death of ∼25% of islet cells following 24- and 48-h incubations (Figure 1C). IL-1-induced islet cell death correlated with an increase in nitric oxide production (Figure 1D), and NMMA attenuated both islet cell death and nitric oxide production. These findings suggest that IL-1 induces islet and RINm5F cell death, that cell death correlates with the production of nitric oxide, and that cell death is attenuated by the inhibition of NOS.
RINm5F cells (A and B) or rat islets (C and D) were treated with IL-1 (10 units/ml) and NMMA (2 mM) for either 24 or 48 h, as indicated. The cells were isolated and viability determined using the MTT assay (A and C). The culture supernatants were isolated and nitrite production was determined using the Griess assay (B and D).
(E and F) The effects of exogenously produced nitric oxide supplied by the donor compound DEANO (500 μM) and the apoptosis inducer camptothecin (25 μM) on RINm5F cell viability (E) and the apoptosis inducer staurosporine on rat islet cell viability (F) were determined by MTT assay.
Cell viability data is expressed as percent death. Results for cell viability and nitrite production are the average ± standard error of the mean (SEM) of three independent experiments. *p < 0.05, significantly different from untreated controls. **p < 0.05, significantly different from IL-1-treated condition.
To examine whether nitric oxide can directly modulate cell viability, the effects of exogenously supplied nitric oxide on RINm5F cell viability were examined. RINm5F cells were chosen for these studies because they represent a homogenous population of β-cells, and the effects of cytokines on these cells have been characterized in detail [9]. Treatment of RINm5F cells for 24 h with the nitric oxide donor compound DEANO resulted in a 25% increase in cell death (Figure 1E). Following a 48-h exposure to DEANO, the level of death was not further increased, a finding that is likely due to the rapid decomposition of DEANO (which has a half-life of ∼2 min under these conditions). The effects of DEANO were compared to known inducers of apoptosis. The topoisomerase inhibitor camptothecin is a classical inducer of apoptosis in rapidly proliferating cell lines [37] and, as shown in Figure 1E, induces the death of ∼40% and 75% of RINm5F cells following 24- and 48-h incubations, respectively. Camptothecin is not an effective inducer of islet cell death (most likely due to the quiescent nature of these cells [unpublished data]); however, the kinase inhibitor staurosporine, another known inducer of apoptosis [38], stimulated the death of ∼40% of islet cells following a 24-h incubation (Figure 1F).
A second assay of cell viability, neutral red uptake, was used to confirm the observations that cytokines induce β-cell death (Figure 2). This assay is based on the ability of viable cells to accumulate neutral red dye, while nonviable cells fail to accumulate this dye [29]. Similar to the results presented for the MTT assay in Figure 1, IL-1 reduced RINm5F cell viability by ∼20%–25% following 24- or 48-h incubations, and cell death in response to this cytokine was attenuated by NMMA (Figure 2A). The apoptosis inducer camptothecin also reduced RINm5F cell viability by ∼60%–80% following 24- and 48-h incubations, as determined by neutral red uptake (Figure 2B). These findings provide evidence, in two different assays of cell death, that IL-1 reduces β-cell viability and that the loss of viability is mediated, in part, by the production of nitric oxide.
RINm5F cells were treated for 24 h or 48 h with IL-1 (10 units/ml), NMMA (2 mM) (A), or camptothecin (25 μM) (B), and the cells were isolated and percentage of death determined using the neutral red uptake assay. Results are average ± SEM of three independent experiments. *p < 0.05, significantly different from untreated controls. **p < 0.05, significantly different from IL-1-treated condition.
Morphological Analysis of IL-1-Induced β-Cell Death
While performing the cell viability experiments, clear morphological differences were observed for RINm5F cells treated with IL-1 as compared to the apoptosis inducer camptothecin. Control, untreated RINm5F cells grew as a flat monolayer of cells with distinct cell borders (Figure 3A). Treatment with IL-1 resulted in morphological changes that included cell swelling and the appearance of holes in the cytoplasm (Figure 3B), changes that are consistent with a necrotic form of cell death. Nitric oxide appears to be responsible for the morphological changes induced by IL-1, as NMMA attenuated RINm5F cell swelling and the formation of cytoplasmic holes (Figure 3C). The morphological changes in RINm5F cells undergoing apoptosis were strikingly different from the alterations induced by IL-1. RINm5F cells that underwent apoptosis following a 24-h treatment with camptothecin were severely shrunken and contained compacted nuclei, consistent with an apoptotic morphology (Figure 3D). This morphological analysis of the effects of IL-1 on RINm5F cell viability suggests that cytokine-induced β-cell death is not caused by classical apoptotic mechanisms and may occur by necrotic mechanisms.
RINm5F cells were treated for 24 h with IL-1 (10 units/ml) (B and C), NMMA (2 mM) (C), or camptothecin (25 μM) (D), and cellular morphology was examined by phase-contrast microscopy. Treatment with IL-1 resulted in cell swelling and detachment (B), events that are prevented by NMMA (C). In contrast to the effects of IL-1, induction of apoptosis using camptothecin (D) resulted in cell shrinkage and condensation of the nucleus. Untreated control cells are shown in (A). Results are representative of three independent experiments.
IL-1 Stimulates β-cell DNA Damage in a Nitric Oxide-Dependent Manner
The TUNEL assay is commonly used to detect DNA damage in cells, and the results have frequently been used as evidence to support an apoptotic pathway of cell death. Using the TUNEL assay, the effects of IL-1 on the integrity of RINm5F and rat islet cell DNA were examined. In this assay, DNA strand breaks were detected by the incorporation of fluorescein dUTP (green fluorescence, Figure 4), and nuclei were visualized by co-staining with DAPI (blue fluorescence, Figure 4) or colocalized with insulin-containing islet cells (using a CY3-conjugated secondary antibody; red fluorescence, Figure 5). Treatment of RINm5F cells for 24 h with IL-1 resulted in DNA damage in ∼50% of the cells (see Figure 4B), and the level of DNA damage increased to ∼65% of the cells following a 48-h incubation (see Figure 4C). Importantly, IL-1-induced DNA damage was attenuated by NMMA to levels similar to those observed in untreated control cells (∼5%–8% TUNEL-positive; see Figure 4E and 4F). Representative fluorescent micrographs indicate that the morphology of TUNEL-positive RINm5F cells following treatment with IL-1 is significantly different from the morphology of cells treated with the apoptosis inducer camptothecin. While TUNEL staining in most RINm5F cells treated with IL-1 appeared uniform throughout the nuclei and colocalized with DAPI staining, there were a number of RINm5F cells in which the TUNEL staining appears to spill out of the nucleus (see Figure 4B and 4C, white arrows), indicating a loss of nuclear membrane integrity. Camptothecin stimulated DNA damage in ∼60% of the RINm5F cells, as shown in Figure 4D (white arrow), and the pattern of TUNEL and DAPI staining was distinctly different from that observed following IL-1 treatment. Camptothecin-induced TUNEL and DAPI staining appears more intense and punctate, indicating that chromatin condensation has occurred.
Treatment of RINm5F cells for 24 h (B) or 48 h (C) with IL-1 (10 units/ml) resulted in DNA damage, as determined by TUNEL staining (green fluorescence). DNA damage was attenuated by NMMA (E and F). The apoptosis inducer camptothecin (25 μM) stimulated DNA damage (D), and this DNA damage is morphologically distinct from that induced by IL-1. Nuclei are stained with DAPI (blue). The morphological differences in TUNEL staining, the loss of nuclear membrane integrity in response to IL-1 (B and C) and the condensation of DNA in response to camptothecin (D) are highlighted using white arrows. The levels of DNA damage in control cells (A) were less than 5%. Results are representative of three independent experiments.
Treatment of rat islet cells for 24 h with IL-1 resulted in DNA damage as determined by TUNEL staining, and ∼95% of this damage occurred in insulin-containing cells (red fluorescence) (B). Nitric oxide appears to mediate IL-1-induced DNA damage, as it was attenuated by the NOS inhibitor NMMA (2 mM) (D). The apoptosis inducer staurosporine (1 μM) also stimulated DNA damage in both insulin containing and non-insulin containing cells (C). Less than 10% of control, untreated islet cells were TUNEL-positive (A). Results are representative of three independent experiments.
Similar to RINm5F cells, treatment of rat islets for 48 h with IL-1 resulted in DNA damage in ∼50% of islet cells, and over 95% of this DNA damage was found in β-cells, as determined by immunolocalization of TUNEL-positive cells with insulin-containing cells (Figure 5B). IL-1-induced DNA damage was prevented by NMMA (Figure 5D), indicating that nitric oxide is primarily responsible for the DNA damage observed in rat islet cells treated with IL-1. IL-1-induced DNA damage was compared with damage caused by the apoptosis inducer staurosporine. Treatment of rat islets for 48 h with staurosporine resulted in TUNEL-positive staining in ∼50% of islet cells, and this DNA damage was observed in both insulin-containing and non-insulin-containing cells (Figure 5C). Similar to RINm5F cells (see Figure 4), the morphology of TUNEL staining in the IL-1-treated islet cells appears uniform throughout the nucleus (Figure 5B, white arrows). In contrast, staurosporine-induced TUNEL staining in islet cells was more intense and punctate (Figure 5C, white arrows). Taken together, these findings suggest that IL-1 stimulates DNA damage in β-cells, and that nitric oxide is a primary mediator of this damage. In addition, these findings indicate that DNA damage induced by IL-1 results in morphological changes in β-cells that are markedly different from the DNA damage observed in β-cells undergoing staurosporine-induced apoptosis.
IL-1 Fails to Activate Caspase-3 in RINm5F Cells or Rat Islets
To directly examine whether IL-1 induces classical apoptosis in rat islets or RINm5F cells, the effects of this cytokine on the activation of caspase-3 were examined. We decided to examine caspase-3 because it is an executioner caspase that is a downstream target of caspases involved in both the intrinsic and extrinsic pathways of apoptosis [39,40]. IL-1 failed to stimulate caspase-3 activation in RINm5F cells (Figure 6A) or rat islets (Figure 6B) treated for either 24 or 48 h. Since nitric oxide is a known inhibitor of caspase activity [41–43], the effects of IL-1 alone and in combination with NMMA on caspase-3 activity were examined. In the presence of NMMA, IL-1 still failed to stimulate caspase-3 activation in RINm5F cells or rat islets. In contrast to the lack of caspase-3 activation in response to IL-1, camptothecin induced a ∼9-fold increase in caspase-3 activity in RINm5F cells (Figure 6A), and staurosporine stimulated a ∼5-fold increase in caspase-3 activity in rat islets (Figure 6B).
(A) IL-1 (10 units/ml), alone or in combination with NMMA (2 mM) and the nitric oxide donor DEANO (500 μM), failed to activate caspase-3 in RINm5F cells following a 24- or 48-h exposure. As a positive control, the apoptosis inducer camptothecin (25 μM) stimulated a 9-fold increase in RINm5F cell caspase-3 activity.
(B) Similar to RINm5F cells, in rat islets IL-1 (10 units/ml) alone or in combination with NMMA (2 mM) failed to activate caspase-3 following a 24- or 48-h incubation. The apoptosis inducer staurosporine (1 μM) stimulated a 5-fold increase in rat islet caspase-3 activity. Results are the average ± SEM of three independent experiments. *p < 0.05, significantly different from untreated control.
Effects of IL-1 on Annexin V Staining in RINm5F Cells and Rat Islets
The externalization of PS on the outer membrane surface of cells is a hallmark of cells undergoing early or intermediate stages of apoptosis that can be quantified by flow cytometry using the calcium-dependent phospholipid-binding protein annexin V [30]. Using this assay, we show that IL-1 fails to stimulate PS externalization on RINm5F cells (Figure 7A) or rat islet cells (Figure 7B). In contrast, ∼55% of RINm5F cells stained positive for annexin V following a 24 or 48-h incubation with camptothecin, and ∼15% of rat islet cells stained positive for annexin V following a 24- or 48-h incubation with staurosporine. The absence of annexin V staining on rat islet cells and RINm5F cells treated with IL-1 is consistent with the lack of caspase-3 activation, and suggests that, while IL-1 stimulates β-cell death, it does not appear to occur by classical apoptotic mechanisms.
Treatment of RINm5F cells (A) or rat islet cells (B) with 10 units/ml IL-1 did not stimulate PS externalization, as determined by annexin V staining and quantified by flow cytometry. In contrast, treatment of RINm5F Cells with camptothecin (25 μM) (A) or rat islet cells with staurosporine (1 μM) (B) stimulated the externalization of PS. Results are presented as percent increase in annexin V-positive cells above the basal levels (∼3%–7%) detected on untreated control RINm5F or rat islet cells and are the average ± SEM of three independent experiments. *p < 0.05, significantly different from untreated control.
Biochemical Evidence that IL-1 Stimulates β-Cell Necrosis
HMGB1 is a chromatin-binding protein that is selectively released by cells undergoing necrosis [26,44–46]. It is not released from cells undergoing apoptosis, as it is sequestered by chromatin [45]. To determine whether IL-1 stimulates β-cell necrosis, RINm5F cells were treated for 24 or 48 h in the presence or absence of NMMA, and the supernatants were isolated and examined by Western blot analysis for the presence of HMGB1. As shown in Figure 8A, IL-1 stimulates the release of low levels of HMGB1 by RINm5F cells, which is first apparent following a 24-h incubation, with higher levels released following a 48-h incubation. In a similar manner, IL-1 stimulated the release of HMGB1 by rat islets (Figure 8B). NMMA attenuates IL-1-induced HMGB1 release by both rat islets and RINm5F cells, indicating that nitric oxide is a mediator of β-cell necrosis. To confirm that nitric oxide induces β-cell necrosis, we show that a 24-h incubation of RINm5F cells (Figure 8C) or rat islets (Figure 8D) with the nitric oxide donor DEANO results in HMGB1 release. In contrast, barely detectable levels of HMGB1 were released by RINm5F cells treated with camptothecin or islets incubated with staurosporine for 24 h (Figures 8C and 8D). As a control, inactivated DEANO failed to stimulate HMGB1 release by either rat islets or RINm5F cells (unpublished data). The release of HMGB1 by rat islets and RINm5F cells provides biochemical evidence that IL-1 stimulates β-cell death by necrotic mechanisms.
(A and B) RINm5F cells (A) or rat islets (B) were treated for 24 or 48 h with 10 units/ml of IL-1 and 2 mM NMMA as indicated. The levels of HMGB1 released into the supernatant (s) and contained in the cells (p) were determined by Western blot analysis. Results show that IL-1 stimulates the release of HMGB1 from RINm5F cells and rat islets, and that this release is attenuated by the NOS inhibitor NMMA.
(C) Treatment of RINm5F cells for 24 h with DEANO (500 μM) stimulated HMGB1 release, while the apoptosis inducer camptothecin (25 μM) failed to stimulate HMGB1 release from RINm5F cells.
(D) Nitric oxide supplied exogenously via treatment with DEANO (500 μM) also stimulated HMGB1 release by rat islets, while HMGB1 was not released under conditions of β-cell apoptosis in response to treatment with staurosporine. Results are representative of three independent experiments.
Effect of Caspase-3 Inhibition on Cytokine-Induced β-Cell Death
To further explore the mechanisms of β-cell death, the effects of caspase-3 inhibition on β-cell death in response to apoptosis inducers and IL-1 were examined. As expected, the caspase-3 inhibitor Z-DQMD-CHO attenuated camptothecin-induced RINm5F cell death, as determined by MTT assay (Figure 9A), annexin V staining (Figure 9B), and caspase-3 activation (unpublished data). In contrast, inhibition of caspase-3 failed to attenuate IL-1-induced RINm5F cell death following a 24- or 48-h incubation (Figure 9C). Furthermore, caspase-3 inhibition failed to prevent RINm5F cell HMGB1 release induced by the exogenous addition of nitric oxide by the donor DEANO (Figure 9D), or following a 48-h incubation with IL-1 (Figure 9E). These findings demonstrate that β-cell death in response to IL-1 treatment does not require the activation of caspase-3, an executioner caspase, and that caspase-3 inhibition does not prevent β-cell necrosis in response to IL-1 treatment or exposure to exogenous nitric oxide, as assessed by the release of HMGB1.
The caspase-3 inhibitor Z-DQMD-CHO (100 μM) attenuated RINm5F cell death induced by a 24-h incubation with camptothecin as determined by MTT assay (A) or annexin V staining (B). This caspase-3 inhibitor failed to prevent RINm5F cell death in response to a 48-h incubation with 10 units/ml IL-1 as determined by the MTT assay (C). Z-DQMD-CHO also did not attenuate HMGB1 release by RINm5F cells treated for 24 h with 500 μM DEANO (D) or for 48 h with 10 units/ml IL-1 (E). The levels of HMGB1 released into the supernatant (s) and contained in the cells (p) were determined by Western blot analysis. Results for MTT assay (A and B) and annexin V staining (C) are the average ± SEM of three independent experiments. *p < 0.05, significantly different from untreated control. **p < 0.05, significantly different from camptothecin-treated condition. Results for HMGB1 release are representative of three independent experiments.
Cytokines Stimulate HMGB1 Release from Human Islets in a Nitric Oxide-Dependent Manner
In contrast to the effects of IL-1 on rat islets, a combination of cytokines (IL-1, TNF, and IFN-γ) is required to stimulate nitric oxide synthase expression and nitric oxide production from human islets. Previous studies have shown that, individually, these cytokines fail to stimulate iNOS expression or nitric oxide production, or to induce human islet cell death [47–49]. To examine whether nitric oxide modulates human islet viability, we examined the effects of a 48-h incubation of human islets with IL-1, IFN-γ, and TNF on the release of HMGB1. Treatment of human islets with this cytokine mixture stimulated a ∼7 fold increase in nitrite production (Figure 10A), and nitric oxide production correlated with high levels of iNOS expression (unpublished observation). Importantly, under conditions in which human islets produce nitric oxide, HMGB1 accumulated in the supernatant of these islet cultures (Figure 10B). HMGB1 release appears to be dependent on the production of nitric oxide, as the iNOS selective inhibitor AG attenuated the production of nitric oxide and the release of HMGB1 by human islets. The apoptosis inducer staurosporine failed to stimulate the production of nitric oxide or release of HMGB1. Alone, neither IL-1, TNF, nor IFN-γ stimulated nitric oxide production, iNOS expression, or the release of HMGB1 from human islets following a 24- or 48-h incubation (unpublished data), providing evidence that the increased production of nitric oxide by human islets in response to the combination of cytokines is required for the release of HMGB1 by human islets. Overall, these findings are consistent with the effects of IL-1 on rat islets and RINm5F cells, in that they provide direct evidence that nitric oxide mediates the release of HMGB1, a marker of necrotic cell death, by human islets in response to cytokine treatment.
Human islets (200 islets in 400 μl of complete CMRL-1066) were treated with a combination of IL-1 (50 units/ml), IFN-γ (500 units/ml), and TNF (50 nM) in the presence or absence of the NOS inhibitor AG (2 mM) or treated with staurosporine (1 μM) alone.
(A) Following 48h -incubation the supernatant was removed and nitrite production was determined using the Griess assay. The levels of HMGB1 released into the supernatant (s) and contained in the islets (p) were determined by Western blot analysis.
(B) Results indicate that cytokine-stimulated HMGB1 release correlates with nitric oxide production, and that inhibition of nitric oxide production using AG attenuates HMGB1 release from human islets. Similar results have been obtained in three of five independent preparations of human islets.
Autoimmune diabetes is characterized by an inability to regulate blood glucose levels due to the autoimmune destruction of insulin-producing pancreatic β-cells. The development of autoimmune diabetes is a T cell-dependent process, as they represent a major cellular constituent found in insulitis, and it is possible to prevent diabetes by T cell depletion [2,3,50]. While T cells are responsible for killing the majority of β-cells during the development of diabetes, relatively little is known concerning the events that initiate this autoimmune response directed against β-cells. Mathis and coworkers [3,51] have recently proposed that β-cell death during normal physiological islet remodeling, which peaks at 14–17 d after birth, may supply β-cell antigens that can then be engulfed by dendritic cells. These antigen-presenting cells (APCs) then ferry the antigen to pancreatic lymph nodes, where they may activate naïve diabetogenic T cells [3,51]. Although the mechanisms of β-cell death during islet remodeling are likely apoptotic, it is possible to mount a T cell response by antigens produced by cells undergoing apoptotic cell death [52].
While experimental evidence supports this model for initiation of diabetes in the NOD mouse [51], this model does not take into account the low concordance rate and variable time of onset of diabetes observed in identical twins [53,54]. Specifically, if β-cell death during normal physiological islet remodeling supplies antigens that activate naïve diabetogenic T cells, one would have to assume that the extent/efficiency of β-cell death and/or antigen processing and presentation to naïve T cells may be highly variable in individuals with an identical genetic makeup. An alternative hypothesis to explain the initiation of autoimmune diabetes, proposed by Lacy [55,56], suggests that intraislet macrophage activation and local release of macrophage-derived inflammatory mediators initiates β-cell death, antigen release, and autoimmunity directed against β-cells. The initiating event leading to intraislet macrophage activation may be viral, bacterial, or chemical in origin. It is well known that viral or bacterial infection stimulates macrophage production of IL-1 and TNF, and these cytokines have been shown to impair β-cell function and induce β-cell death [4,5,57]. In support of this model, previous studies have shown that feeding rodents a diet deficient in essential free fatty acids, known to deplete resident macrophages [58,59], attenuates diabetes induced by the chemical toxin streptozotocin [60] and the natural progression of diabetes in BB rats [61]. In addition, a number of studies have shown that the local release of IL-1 by activated intraislet macrophages in rodent and human islets leads to β-cell dysfunction and damage [13,14,62].
In this report we have evaluated the cellular mechanisms by which the macrophage-derived cytokine IL-1 kills β-cells. Using three different methods to evaluate cell viability, MTT oxidation, neutral red uptake, and TUNEL staining (DNA damage), we show that IL-1 induces β-cell death (see Figures 1–3). Nitric oxide appears to be one mediator of β-cell death, as NOS inhibition attenuated cell death in response to IL-1, and nitric oxide, supplied exogenously using donor compounds, reduced β-cell viability. The mechanism by which IL-1 stimulates β-cell death is both morphologically and biochemically different from known inducers of apoptosis. Morphologically, IL-1-induced β-cell death is characterized by cell swelling and diffuse TUNEL staining–changes indicative of the loss of membrane integrity, or necrosis. In response to inducers of apoptosis, the morphology of dying β-cells differs drastically from the effects of IL-1. Apoptosis induction results in cell shrinkage and tight, punctate TUNEL staining, consistent with the form of cell death that we have observed with known inducers of apoptosis. To investigate biochemical mechanisms responsible for β-cell death, we evaluated the effects of IL-1 on caspase-3 activation and PS externalization (annexin staining). Caspase-3 activity was chosen because it the executioner caspase activated by initiator caspases involved in both extrinsic and intrinsic apoptotic pathways [39,40]. While many reports have suggested that IL-1 induces β-cell death by apoptosis, we show that IL-1 fails to activate caspase-3 or to stimulate annexin V staining in islets or RINm5F cells. In contrast, known apoptosis inducers stimulated 6- to 9-fold increases in caspase-3 activity and significantly increased annexin V staining on RINm5F cells and rat islet cells. The lack of RINm5F cell and islet caspase activity following treatment with IL-1 is not surprising, as IL-1 stimulates β-cell production of nitric oxide, and nitric oxide has been shown to inhibit caspase activity by S-nitrosation of the active site cysteine [41,43].
Since these findings suggest that the macrophage-derived cytokine IL-1 does not induce apoptosis, we sought to identify whether IL-1 kills β-cells by necrosis. The morphological changes (see Figure 3) and the diffuse TUNEL staining in RINm5F cells and rat islet cells (see Figures 4 and 5) induced by IL-1 treatment are consistent with a necrotic type of cell death. To provide biochemical evidence in support of IL-1-induced β-cell necrosis, the effects of this cytokine on the release of HMGB1 from β-cells was examined. HMGB1 is a chromatin-binding protein that functions to distort the double helix, allowing proper physical interactions between transcription factors and chromatin [63]. Recent studies have shown that HMGB1 is released from cells dying by necrosis, but it remains tightly bound to chromatin in cells undergoing apoptosis [26,44–46]. In this study, we show that IL-1 stimulates HMGB1 release from RINm5F cells and rat islets in a nitric oxide-dependent manner, and that HMGB1 release is not affected by the inhibition of caspase activity. These findings suggest that IL-1-induced HMGB1 release is mediated, at least in part, by nitric oxide, a conclusion further supported by the ability of nitric oxide to directly stimulate HMGB1 release by RINm5F cells and rat islets. Importantly, under conditions in which RINm5F cells or islets have been forced to undergo apoptosis, HMGB1 was not released. Overall, this morphological and biochemical evidence supports β-cell necrosis as the primary mechanism by which IL-1 induces β-cell death, and that this death pathway is mediated, in part, by nitric oxide.
While IL-1 is sufficient to inhibit the function and to induce the death of rat β-cells, alone, the exogenous addition of IL-1 does not inhibit the function or induce death of human β-cells [47,48]. A combination of the cytokines IL-1, TNF, and IFN-γ is required to impair human β-cell function and induce islet cell death [47,48]. Treatment of human islets for 24- or 48-h with IL-1 + IFN-γ + TNF results in inhibition of glucose-stimulated insulin secretion and mitochondrial aconitase activity, effects attenuated by the iNOS inhibitors NMMA and AG [47,64]. In addition, the local release of IL-1 by intraislet macrophages in response to TNF + IFN-γ + LPS treatment results in inhibition of glucose-stimulated insulin secretion that is attenuated by inhibitors of iNOS [14]. These findings support a role for nitric oxide as a mediator of cytokine-induced β-cell damage in human following exposures of 24 and 48 h. In contrast, extended incubation (for 6–9 d) of human islets with IL-1 + TNF + IFN-γ has been reported to inhibit insulin secretion and induce β-cell apoptosis in a nitric oxide-independent manner [19,48]. The latter findings have led to speculation that human islets are less sensitive to nitric oxide than are rodent islets [65]. Comparisons of cellular responses to cytokines across species are complicated by the genetic background of each species. This is especially true when using human islets in which the genetic background is different for each individual human islet preparation. In contrast, studies with rodent islets are usually performed with a specific strain of rat or mouse. Importantly, Nerup and coworkers have shown that the ability of IL-1 to inhibit β-cell function and to induce iNOS expression varies in three different rat strains [66]. These findings suggest that the genetic background, even within a given species of animal, contributes to the islet response to cytokines. Taking this into account, we have evaluated the effects of IL-1, TNF, and IFN-γ on nitric oxide production and HMGB1 release by human islets. In three of five preparations, the combination of cytokines stimulated HMGB1 release by human islets by a mechanism that was sensitive to inhibition by the iNOS inhibitors AG (Figure 10) and NMMA (unpublished data). Importantly, in two human islet preparations the combination of cytokines failed to stimulate HMGB1 release and also failed to stimulate nitric oxide production or iNOS expression. The reason for this lack of response to cytokines in two of the human islet preparations is unknown; however, it has been reported that elevated levels of heat-shock protein 70 attenuate the ability of β-cells to express iNOS and produce nitric oxide, and the strain differences in response of rat islets to IL-1 have been reported to be associated with differences in levels of heat-shock protein 70 expression [67–69]. Taking this into account, our findings suggest that when human islets produce sufficient levels of nitric oxide in response to cytokines, one consequence is the release of HMGB1. In contrast, when human islets fail to respond to cytokines by producing nitric oxide, they also do not release HMGB1.
HMGB1 is not only a marker of cells undergoing necrosis, but it is also immunologically active. It has been shown to bind to Toll2 and Toll4 receptors and to activate nuclear factor κB-dependent gene expression [70–72], and antagonists of HMGB1 have been shown to attenuate sepsis in animal models [73]. It is well established that necrotic cells release immunological adjuvants that activate APCs and that amplify and sustain T cell-dependent immune responses [74–76]. Recently, Rovere-Querini et al. showed that necrotic HMGB1-deficient cells have a reduced capacity to activate APCs, and that HMGB1 neutralization attenuates the ability of supernatants derived from necrotic wild-type cells to activate APCs [77]. HMGB1 also appears to enhance primary antibody responses to soluble antigens and to enhance cytotoxic lymphocyte responses [77]. While caution should be exercised when extrapolating in vitro findings to mechanisms of disease pathogenesis, it is tempting to speculate on the potential role of intraislet macrophage activation as a precipitating event in the development of autoimmune diabetes. In previous studies, we have shown that the 10–15 resident macrophages found in islets, when activated, produce IL-1 to levels sufficient to inhibit β-cell function in a manner similar to that occurring with the exogenous addition of this cytokine to islets [13,14,78]. We now show that, at these same concentrations, IL-1 kills β-cells by necrotic mechanisms that are associated with the release of the immunological adjuvant HMGB1–a molecule known to augment APC activation and cytolytic responses of T cells.
We would like to thank Colleen Bratcher for expert technical assistance. National Institutes of Health Grant DK 52194 and DK 68839 (JAC) and predoctoral fellowship awards from the American Heart Association (SAS and KTC) supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions
SAS, ALS, KTC, and JAC designed the study, analyzed the data, and contributed to writing the paper.
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Patient Summary
Type 1 diabetes (also called autoimmune diabetes or juvenile diabetes) is an autoimmune disease. For unknown reasons, at some point in childhood or adolescence, the body's own immune system starts attacking and destroying the insulin-producing islet cells in the pancreas. Once the majority of islet cells are destroyed, patients can no longer produce insulin to regulate their blood sugar and must depend on strict diets and insulin injections. Scientists are trying to understand the early events during the development of the disease. There are two fundamentally different kinds of cell death in cells of higher animals and humans, called apoptosis and necrosis. Apoptosis (also called programmed cell death) is an organized, clean way in which cells die without spilling their contents and without causing an inflammatory immune response. They are simply gobbled up by other cells that serve as the body's garbage collectors. Necrosis, on the other hand, is a more messy process and one that does activate the immune system and cause local inflammation.
Why Was This Study Done?
The scientists who did this study are interested in the early stages of islet cell death. Specifically, they wanted to know whether islet cells during the early events of autoimmune diabetes die via apoptosis or necrosis. Earlier experiments to address this question had yielded no clear-cut results.
What Did the Researchers Do and Find?
All the experiments for this study were done in cultured cells in the laboratory. For the most part, the researchers used rodent islet cells, and then they confirmed the crucial finding in human islet cells. They grew the cells under conditions that resembled, to the best of their knowledge, the early stages of diabetes, which caused some of the cells to die. They then did a variety of tests to see whether that cell death was through apoptosis or necrosis, and the results showed that the latter was the case. They also identified some of the key factors involved in promoting and executing the necrosis process.
What Does This Mean?
One must always be careful to extrapolate from laboratory results like these. With this caveat, the results suggest that early in the development of diabetes cells die by necrosis, and they point to some of the key factors involved. These are important results that will inform future studies toward the goal of understanding autoimmune diabetes well enough to prevent or stop its development.
Where Can I Find More Information Online?
The following Web sites provide information on autoimmune diabetes.
MedlinePlus pages on type 1 diabetes:.
Web site of the Juvenile Diabetes Research Foundation:.
Pages on type 1 diabetes from the Canadian Diabetes Association:.
Type 1 diabetes pages from the UK National Institute for Health and Clinical Excellence:.
UK National Diabetes Information Clearinghouse:.
American Diabetes Association Web site:.


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The Holy Land remains a cachet entry on many a bucket list. You would be hard pressed to find any other place quite like Jerusalem.
Imbued with the history and reverence of three world religions, Jerusalem is a spiritual cradle of truly biblical proportions. No other city can boast such significance to Judaism, Christianity and Islam.
For the first-time visitor, the sheer range of the historic treasures is not just a head rush but potentially hard to navigate.
The life and times of Jesus Christ capture prime interest, and I recommend you concentrate your sightseeing on that. Irrespective of your beliefs, the faithful and the agnostic alike will be inspired by the sights.
The Via Dolorosa is the ultimate walking tour, retracing the path Jesus is believed to have taken, along with his cross, to his crucifixion at Calvary.
The Via Dolorosa, which means Way of the Sorrows, begins in the Muslim quarter of the Old City, a multi-sensory riot of market life. The Stations of the Cross are marked out along the route, culminating in the site of his death and entombment.
The Church of the Holy Sepulchre is the holiest Christian site in Jerusalem, and pilgrims pour in to experience where Christ spent his last hours.
Built on top of Golgotha in 326AD, the church is governed by various Christian denominations including Catholic, Orthodox and Armenian. A Greek Orthodox chapel has its altar directly over the rocky outcrop on which Christ was nailed to the cross. Below the altar, you can bend down and touch the rock into which the cross was hammered.
A piece of Golgotha rock can be seen inside the church, shielded by protective glass. The crack in the rock is believed to have been caused by the violent earthquake which hit immediately after Christ's death.
In thefirst century, Calvary was a disused stone quarry on the fringe of the Old City. Tombs were commonly cut into the rock, including the tomb where Christ was laid.
East of the Old City, the Mount of Olives delivers picture-perfect vistas. Hugely popular is the Garden of Gethsemane and the Church of All Nations. Venerating the site where Christ prayed to God the night before he was arrested, the garden features the last surviving olive trees from 2000 years ago.
A short drive from Jerusalem takes you to Palestinian-controlled Bethlehem, where you can explore the gorgeous Church of the Nativity and shop for an exquisite nativity scene made from olive-tree wood.

subset: commoncrawl


text:
Reebok Skyscape Sneaker - Womens
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Item # 299448
With modern updates and a lightweight design, your feet will be over-the-moon happy when you are wearing the women's Reebok Skyscape sneakers! These cool low-top lace-ups have a super soft fabric upper and cushioning right where you need it.
- Fabric upper
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- Lightly padded collar and tongue for comfort
- Soft fabric lining
- Cushioned footbed
- Molded EVA outsole provides lightweight cushioning, durability and enhanced flexibility
- Imported
- View more Women's Reebok Shoes
By: Boogalue
Location: Atlanta
June 07, 2014
Feels like walking on clouds
"I fractured my ankle and my doctor told me to wear sneakers for four months after he removed my cast. These shoes were the perfect fit for my rehab. These shoes are soft all over and are great to walk in at work. Plus they aren't too flashy like some sneakers can be. I just need them in a few more colors, maybe red or blue. I'll certainly buy them again!"
By: FrankieRiley
Location: MN
May 20, 2014
My Favorite!
"I've had these for about three weeks and I can say without a doubt that they are the most comfortable shoes I've ever worn. They are very light weight and feel like a padded bra. I wear them all day. I have wide feet and every shoe I've tried has given me a blister after an hour of walking. Not these! Sometimes I wear socks, sometimes I don't. The soles stay pretty clean. I don't feel the need to take them off just to run in the house, even on carpet. I dropped some hair dye on the top and didn't notice until the next morning and it washed right off the foam and laces with just a little water. I did get into a bit of mud and had to machine wash them (the insoles pop right out). They didn't come out looking brand new; the edges are a little dark. I washed them with some clothes though. A crease has formed across the toe, I know that bothers some people. I'm always checking DSW to see if they added more colors. I will definitely be buying more. I love these shoes!"
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Doug and Laurel Weaver of Castle Dale announce the marriage of their daughter Shalise to Daniel Lee Cook, son of Chall and Annette Cook of Huntington. They will be married on March 4 in the Manti LDS Temple. A reception will be held in their honor for all family and friends the following evening, March 5, from 6-8 p.m. at the Senior Citizen Center, 85 North 200 East in Castle Dale.
Shalise and Dan are both 2001 graduates of Emery High School. Shalise graduated with an associate degree from College of Eastern Utah in 2003. She spent the past year working as a nanny in New York and is currently enrolled in the physical therapy program at Salt Lake Community College.
Dan served a LDS mission in the Quito Ecuador Mission. He has since completed his associate degree at CEU and is currently employed at the Gateway Center in Salt Lake City.
The couple will reside in Salt Lake City where both plan to continue their education.
Tom and Loyette Holdaway, and Bruce Myerhoff, announce the marriage of their daughter Heather Noelle Myerhoff to Charles Max Wood, son of Dr. Max and Jackie Wood of Orem. They will be married in the Manti Temple on March 4.
The couple will be honored with a reception at the Huntington Stake Center that evening from 6-8 p.m. An open house will follow the next week.
Chuck and Heather will make their home in Provo.


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Top Definition
One of the most notorious kids in all of Cranford. The holder of the Palooza. Supers powers include many women being attracted to him, an oversized penis which rivals that of a humpback whale, super srength, and ultimate coolness. His garage (where various Snakeapaloozas are held) is named the Snake pit. His only weakness is old neighbors...one call to the police and his powers ar rendered useless.
"Wow, is that Jake the Snake? What a good looking man...maybe he'll have my children."
by asdfdf September 04, 2006
The Urban Dictionary Mug
One side has the word, one side has the definition. Microwave and dishwasher safe. Lotsa space for your liquids.Buy the mug
The Urban Dictionary T-Shirt
Smooth, soft, slim fit American Apparel shirt. Custom printed. 100% fine jersey cotton, except for heather grey (90% cotton).Buy the t-shirt


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Clairol The Uncolor System Haircolor Remover
- Overview
- Ingredients
- Features
- Removes semi-permanent and temporary colors
- Removes semi-permanent transparent hair colors
- Removes the build-up left by styling aids
- Leaves natural haircolor untouched
- Leaves hair soft, resilient and ready for re-coloring
- Apply Uncolor to dry hair directly from applicator bottle.
- Work up lather, making sure all hair is covered.
- Wrap hair in plastic cap.
- Place under pre-heated dryer for 30 minutes.
- Rinse, shampoo and condition.
- Hair is now ready to color.
- Repeat step 1 and 2 above.
- Leave on for five minutes.
- Rinse, shampoo and condition.
- About the brand
Water (Aqua), Lauramide DEA, Sodium Lauroyl Sarcosinate, TetrahydroxypropylEthylenediamine, Coco-Betaine, DMDM Hydantoin, Fragrance, Methylparaben,Propylparaben, Sodium Sulfite, Tetrasodium EDTA.
To remove styling aids build-up from permanently colored or natural colorhair:
Today, Clairol is a part of Procter & Gamble, but it was founded in 1931 by an American chemist and his wife, Lawrence and Joan Gelb. The found inspiration for their company's name while traveling in France: Clairol is the name of a hair coloring preparation they discovered there. Clairol started off making hair color for salons, but in 1950 Clairol revolutionized hair care by bringing trusted and easy to use coloring home. Clairol has 6 lines: nice 'n easy, PERFECT 10, natural instincts, Herbal essences color, BALSAM, and Hydrience.


text:
Audrey Brooke Estella Pump
Sorry, this item is sold out!
Looks like you've got more shoe shopping to do - have fun!
Looks like you've got more shoe shopping to do - have fun!
Item # 209776
Ruffles are all the rage this season. Slip on the Audrey Brooke Estella peep-toe pump for your next special event to feel girly and glamorous.
- Satin upper with ruffled T-strap
- Hidden platform
- Adjustable ankle strap
- ¼" platform, 4¼" satin covered heel
- Synthetic sole
- Imported
By: Hokiegirl525
Location: Virginia
January 13, 2013
"These shoes are very comfortable and elegant. I bought these shoes in white to match my wedding dress. What I like most about the shoes is that they are a good height that I feel like will be comfortable to wear all night long. I also like that they are simple, yet decorative enough that they don't overpower my dress! I am very excited to wear these and would definitely recommend them for other brides!"
By: Villebrun0000000000
Location: MN
December 03, 2012
Cute, But...
"I fell in love with these shoes ONLINE....I igonored the reviews stating they were too narrow and that they run long.I should've listened. I put them on, and they were just uncomfortable and too big. I should've ordered a 1/2 size down. I will keep searching for christmat party heels.. :("
By: Maverick718
Location: Online
October 24, 2012
Wedding approved
"I wore these heals probably over 12 hours as a bridesmaid (minus a couple of take-offs for dancing) and the comfort lasted all night long! I was amazed how I didn't mind being in them so long. And, the style is sleek and eye-catching. The little black heal is always a classic."
By: Starrfire32099
Location: California
October 12, 2012
"I ordered these shoes in ivory for a cruise dinner.They where cute but so narrow that a good portion of my foot hung over the side.I literally started crying when my foot didn't fit! If you have wide feet then do not order these shoes no matter how cute."
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text: Beware of Invention Promoter and Private IP Registration Service Scams By Gibbons P.C. on August 9, 2011 Posted in Patent While invention promoters and IP registration firms claim to assist present and future IP holders, some have been found to offer little or nothing of value in exchange for the thousands of dollars paid to them. Here are ways to investigate these firms and learn about your rights to avoid being treated unfairly.… Continue Reading


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By PATSY STODDARDStaff, Emery County Progress
The boys and girls basketball teams from Emery and Carbon counties battled it out at San Rafael Junior High in Ferron on Feb. 20 and 21.
In the first game the San Rafael girls defeated the Canyon View girls by a score of 45-27. The next game had the Canyon View boys pitted against the Mont Harmon pirates.
The Canyon View team led at the half by a score of 22-14. The second half was a fast paced game with a lot of players getting in foul trouble. The game ended with CVJH 64 and Mont Harmon 53.
The next matchup was the Helper girls against the Mont Harmon girls this battle ended with Mont Harmon defeating the Helper team.
The boys from San Rafael and the Helper Rams were next out on the floor and their contest ended with the Rams on top.
The championship girls game was played at 5 p.m. on Thursday with Mont Harmon and San Rafael going head to head. The score was 25-16 at half time. The SRJH girls played some tough defense to hold off the Pirates. But late in the third quarter the Pirates came alive and the score was SRJH 26 and MHJH 23 with 3:16 left in the quarter. SRJH put in two foul shots to bring their total to 28. San Rafael's Alexis Swasey fouled a Pirate which brought them to the line for two foul shots which they made bringing them to within one point of the Bulldogs.
On the other end of the court Swasey was fouled and sank two foul shots to bring the score to SRJH 30, Mont Harmon 27 with 1:30 left to play in the third. On their next possession Mont Harmon turned over the ball on a traveling violation. Two quick fouls on San Rafael players Swasey and Callie Seely put Mont Harmon at the line. But the free shots went astray. Mont Harmon had a reaching foul which brought Adrian McNee to the line. She missed her foul shot and the ensuing scuffle for the ball brought a jump ball call. San Rafael came up with the ball but stepped on the line giving the ball back to Mont Harmon.
The San Rafael girls kept full court pressure on the team from Price which led to much frustration on their part. McNee fouled a Pirate but they couldn't make their free shot and the score stood at SRJH 30, Mont Harmon 27 at the end of the third quarter. Into the fourth quarter the play was just as intense with McNee going to the line for two foul shots.
Her attempts failed and the ball returned to the Mont Harmon side of the court, but not for long as the ball was stolen away by Natalee Hanson and brought back down the court.
Tahna Merrill of SRJH got in on the fouling which brought a Pirate to the line. She stepped over the free throw line however and the ball returned to San Rafael possession.
A traveling violation sent the ball back to Mont Harmon who in turn traveled with it. The ball was stolen from SRJH and taken down court by the Pirate but it would not drop. San Rafael attempted a three that would not drop.
Tought play continued but at the sound of the buzzerSan Rafael was still on top with a score of 37-30.
The SRJH girls were presented with two trophies, one for the championship and one for tying for first in league play.


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2nd Annual Pursuit of Operational Excellence (OpEx) Conference
October 10-11, 2013
The Systems Science and Industrial Engineering (SSIE) Department of the Watson School of Engineering and Applied Science at Binghamton University proudly presents its second annual Pursuit of Operational Excellence event. This Conference will facilitate improved associations between industry professionals, the SSIE Department, and SSIE students. Industry professionals will leave this Conference with better insights as to how industrial and systems engineering can optimize their operations.
The Conference will highlight the potential for utilizing the full capabilities of industrial and systems engineering practice offered by the SSIE Department and student body, providing case examples.
The 2nd Annual Pursuit of Operational Excellence Conference will be held in conjunction with Alumni Weekend and will feature speakers from industry, professors, and alumni from the SSIE Department. Presentations and discussions will be held in the areas of healthcare systems, energy, and industrial solutions. Participants will gain knowledge on how industrial engineers can lead to simplifying complex systems and can contribute to the success of an organization.
This Conference is co-sponsored with Binghamton University's IIE Student Chapter and Alpha Pi Mu..
Featured Speaker
Rick Duha
- Continuous Improvement Leadership
- Strategic Thinking
- Lean Manufacturing
Who Should Attend
This event is encouraged for a broad spectrum of technical and managerial backgrounds.
Among those who can truly benefit from attending the event are:
- Managers and engineering support personnel
- Industry, government, hospital, military, and university personnel
- All those involved in hiring from the technical pool of college graduates
- Binghamton University Alumni
The event is offered in conjunction with:
Lockheed Martin
BAE Systems
Cook Brothers Companies
XONITEK Consulting Group Int'l LLC
Hidden Valley Electronics
Johnson Outdoors
Sanmina-SCI Owego
Innovation Associates
Raymond Corporation
Broome County Industrial Dev. Agency
PB Industries
United Health Services
Endicott Interconnect


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Skip to comments.Teen Who Shot Baby Dead In Stroller Now Blames Babys Parents For The Murder
Posted on 08/18/2013 8:51:15 AM PDT by barmag25
It was a shocking story from March of this year: DeMarquis Elkins, 17 at the time, deliberately murdered 13-month-old Antonio Santiago in his stroller, because Antonios mother carried no money when Elkins and an accomplice tried to rob her. Now, that tragic story has taken a turn for the bizarre. Elkins, who is being tried with his accomplice, Dominque Lang, 15, claims that Santiagos mother and father put out a contract on their son in order to get insurance money.
On March 21st, his last day alive, little Antonio could toddle but not yet talk. His mother, Sherry West, strapped him in a stroller and took him for a walk near their apartment in Brunswick, Georgia. According to the story she told police at the time, 17-year-old Elkins and a 14-year-old companion suddenly confronted her, waving a gun, and demanding money. West cried as she told the Associated Press how Elkins deliberately slaughtered her child because he viewed it as the reason she had no money to give to him:
He asked me for money and I said I didnt have it, she said. When you have a baby, you spend all your money on babies. Theyre expensive. And he kept asking and I just said I dont have it. And he said, Do you want me to kill your baby? And I said, No, dont kill my baby!
(Excerpt) Read more at mrconservative.com ...
Just. Kill. Him.
Very slowly.
Very slowly.
Quickly - before some limp-wristed judge steps in. Slow would be satisfying, but getting it done is what matters.
It just does'nt seem adequate!
Well,I can't argue with that logic.Put him down.
Well,I can't argue with that logic.Put him down.
Let him wander into the gen pop when he turns 18. I'll bet the Aryan Nations gangs won't even have to lift a finger.
Thugs are expensive. Kill them.
Or Chris Rock's.
ahaha NOW that you Mention it Yes the perp does have an uncanny resemblance to "mister rocks".
we need to see his birth certificate....../s
Too late - some limp-wristed judge (five, actually) has already stepped-in; in this country it's illegal to execute those convicted of even the most heinous of crimes committed prior to reaching their 18th birthday.
I do remember the daughter mentioning that the mother talked about some insurance policy and wondered how much she could get.
If Obama hada son...
"Elkins, who is being tried with his accomplice, Dominque Lang, 15, claims that Santiagos mother and father put out a contract on their son in order to get insurance money."
"Elkins wasnt arrested at the crime scene, but West [the mother] did pick him out of a photo line-up."
So, we're supposed to believe that after putting out a contract on her son, the mother identified the killer which would mean she didn't expect him to reveal the contract. Possible, if she's crazy and stupid, but not likely.
This is caused by Entitlement Mentality.
In his mind, since the mother and child did not give him what he wanted, they were stealing from him.
Reminds me.... Got to go work on my tree chipper.
Stay safe Bill !
Drop him into a woodchiper
And people wonder why it is you might get a little nervous seeing a character like this walking 'round your neighborhood at night in the rain.
Precisely. People who haven't been fingered, don't pick their accomplices out of a lineup. Not even a stupid perp would do that.
If there was a serious amount of insurance put on the baby, the police would have raised some eyebrows. If there were any at all, I'm guessing it was a small whole-life policy that some people get to ensure coverage later in life. As a baby, my grandparents put on on me. I cashed it out in my mid-20s, when I purchased a much ore substantial policy.
Either way he killed the baby so he should get the max. Why would being a hitman instead of a garden-variety punk make any difference?
feet first
BTW, like in Trayvon case: "Was the boy unarmed," hmmm???
In his mind, since the mother and child did not give him what he wanted, they were stealing from him.
Most unfortunately, UnwashedPeasant, that's the crux of the matter. That attitude predominates all the way from the street corner narcotics gangs to the office of the attorney general of the United States.
Welcome to post-constitutional America.
Bailiff take him to the killing room.
Christopher Milke was murdered for a $5000 insurance policy. Roger Scott, one of three convicted in his murder, participated for a promise of $250.
Blowtorch, pliers and a razor blade.
There is a "Saving DeMarquis Elkins" facebook etc just like there is for the Islamic terrorist Boston bomber
There is a "Saving DeMarquis Elkins" facebook etc just like there is for the Islamic terrorist Boston bomber
Give him to "VLAD THE IMPALESR!"
They don't appear we'll off. Could have needed the money for the funeral.
Just maybe the mother needed the insurance money to pay for his funeral~
Planned Parenthood could spin this as an "post-partem abortion".
There must already be a word out there to describe the state of mind of such a person.
Heres Money-ville's Facebook page.
This is obviously the lawyers idea as this guy is too stupid to come up with this defense.
The lawyer knows the little POS is going away for a long time and will try anything.
He needs to be disbarred for being a total POS like his client.
The parents in this situation seem extremely peculiar and shady to me. But even if they did contract a hit on their child, the accused is the one who did it. Which makes it premeditated. Which means that he should never get out of jail.
what a bunch of barbarians.
Woman: "But we have to free Hat.
Stan: It's just that, you know, he killed twenty-three babies.
Man 4: Well yeah, but it was in self-defense!
Crowd: YEAH!
Cartman: He... killed... twenty-three babies in self-defense?
Skeeter: Hat was attacked maliciously and unprovoked by a gang of babies in West Town Park. When that many babies get together they can be like piranha.
Man 5: Three eyewitnesses testified that if Hat hadn't killed those babies, they'd have killed him!
Crowd: YEAH! [the signs go up] Free Hat! Free Hat! Free Hat!"
- South Park, Free Hat episode.
Can't even get original with their stupidity so the have to rip off South Park.
The article said she was concerned about how "soon", not how "much", since the policy would already have been written for a given amount. Isn't it remotely possible that she was concerned about hospital bills for her own shot leg and funeral expenses for the baby? Many people are living at the edge of financial ruin in this economy.
He should be shot while "escaping".
The baby was obviously racist.
I don't know who is more sick, The feral animals who perpetuate this crap or the white Borg who allow it to happen.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


text:
Lagerfeld's Choupette Collection, Miranda and Orlando Split
Your style news update...
Mee-ow—Karl Lagerfeld's famously beloved cat, Choupette, is now the focus of his newest accessories collection, according to WWD. The capsule collection includes a lack and white cat-themed cap, iPhone cover, gloves, a scarf, a cross-body bag and a tote. The pieces will be exclusively available at Karl Lagerfeld stores internationally starting November 12th. Whiskers included.
-And in sad news, Miranda Kerr and Orlando Bloom have called it quits after 3 years of marriage and six years together. The couple has one child together, Flynn and said in a statement, that they "have been amicably separated for the past few months," and, " have recently decided to formalize their separation. [EOnline.com]
-Paris-based fan favorite Carven will open its first US store in Soho. The boutique will be at 83 Mercer Street, and it will be the brand's 16th global location. [WWD]
-Olivia Wilde is taking her first dive into the design world. The actress recently redesigned a Yoana Baraschi dress sold at Anthropologie for her charity, Conscious Commerce. Profits from the dress's sales will be used to fund a girls' school in Calcutta, India. Chic, talented, and philanthropic too—we're more than a little jealous. [WWD]
-Mega-popular rapper Drake has announced that he will exclusively dress in Calvin Klein Collection for his upcoming North American tour, where he'll be performing in 39 cities. [WWD]
-With the help of vintage designer Dr. Romanelli, Coca-Cola has released a 200-piece clothing line. The collection is mainly streetwear, and was inspired by vintage Coke-branded clothing that Romanelli found in flea markets and vintage stores [HuffPost Style]
-Designer Nate Lowman has collaborated with classic tough-kid shoe brand Converse to produce a new collection inspired by Willem de Kooning's 1954 portrait of Marilyn Monroe. These Chucks manage to keep it classy and cool [JustOneEye.com]


text:
Sometimes we look at Mary-Kate and Ashley Olsen's ensembles and we think: Wow, did they get that out of Ozzy Osbourne's closet? Who knew two pint-sized child stars could have so much sartorial overlap with the Prince of Darkness? So, we put together a guessing game. The challenge is at hand: can you tell the difference between Ozzy and Olsen accessories?
Crushable Guessing Game: Olsen Twin or Ozzy Osbourne?
- 6 years ago by Liana Maeby
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Manga reading list: Bleach, Naruto, Katekyo Hitman Reborn, One Piece, Code Breaker, Gamaran, Kuroko No Basket, Fairy Tail, The World God Only Knows, Kimi No Iru Machi, Good Ending, Medaka Box, Nisekoi, D.Gray-Man, Ao No Exorcist, Freezing, Magi - Labyrinth of Magic, Soul Eater, Rosario Vampire Season II, Iris Zero, Ubell Blatt, Bloody Cross, Sun-Ken Rock, and Infinite Stratos,
didn't really like this chapter the teacher, this was annoying; this entire chapter gave me deja vu
Just realized sumtin. Wolverine and Chun Li were in the cover page .
I'm kind of hoping for a scene where Toujou meets jabberwock and he tells him he's only looking for Zenjūrō disciple then toujou levels him and says something badass about him learning under Zenjūrō, with of course jabberwock realizing he's the real deal
Hey baby, wanna take a gander at some Adam West penis?
For shame. That part got me too though lol.
Hey baby, wanna take a gander at some Adam West penis?
The fuck just happened? Something is missing between the end of 123 and 124. The creator has a massive case of ADD.
I Love You!


text:
AK Anne Klein Saisha Croc Wedge Pump
Sorry, this item is sold out!
Looks like you've got more shoe shopping to do - have fun!
Looks like you've got more shoe shopping to do - have fun!
Item # 249370
Update your classic pump with the Saisha by AK Anne Klein Sport. This wedge pump is comfortable and will quickly become one of your favorites.
- Faux leather upper
- Faux patent leather cap toe and heel cover
- Signature embossed bit detail
- Cushioned insole
- 1½" molded heel
- Synthetic sole
- Imported
- View more AK Anne Klein
By: RK555
Location: Baltimore MD
August 25, 2014
"I loved these shoes when I got them from the store but they make this horrible squeaky noise, my whole office could hear me walking around. Couldn't wear them for more than 30 minutes.. Sadly had to return them.. IF you wont be wearing these work or a quite place then buy them :) Else think twice..."
By: Cyndis
Location: Greensboro, NC
August 22, 2014
Love this wedge
"This is a really cute shoe for work, it looks great with all the fall colors because of the black & brown in the shoe. Gives a little height but not too much. Perfect height for my petite pants."
By: Adeline1
Location: Bayside
July 11, 2014
My cute shoes
"I love my new Anne Klein shoes & have received multiple complements on them. Great shoe!!"
By: vln90
March 25, 2014
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Ananta Samakhom Throne Hall HDR
I thought I'd re-edit the Ananta Samakhom Throne Hall photo in HDR.
I thought I'd re-edit the Ananta Samakhom Throne Hall photo in HDR.
The Democracy Monument is a public monument in the centre of Bangkok. It sits on a roundabout on the wide east-west Ratchadamnoen Klang Road, at the intersection of Dinso Road. The monument is supposed to be roughly halfway between Sanam […]
As you may have noticed, this is an almost identical shot of last nights post. Well, this was taken the following day, in the morning. We had a cloudy day in Bangkok, which made for a more dramatic scene. I've processed […]
Asif is a professional photographer shooting weddings, portraits and personal/commercial events throughout the UK and overseas. Asif specialises in portrait and reportage and his style of photography is fresh, creative and always unique. Contact Asif to see how he can help you bring your wedding, portrait portfolio or events vision to life.
Please do not use these images on websites, blogs or other media without my explicit permission.


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Review: goods arrived 2 days later then
goods arrived 2 days later then estimated, but overall good service.
23 Jun 2011 by Liyenita W.
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goods arrived 2 days later then estimated, but overall good service.
23 Jun 2011 by Liyenita W.
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Got what I wanted, when I wanted it cheaper than I hd initially expected. Read more
Matthew Lawrence
Fridge hinge ordered online. No problem and arrived quickly Read more
Great service, good value for money. Order arrived within 2days. Have used before and would Read more
My request for information was answered promptly, based on which I placed my order for replacement Read more
M J Rogers
Very pleased with quality of products and great value Read more
Patricia Colville-Hyde
Great parts, fitted well and arrived really quickly, will use again. Read more
Andrew Thetford
Will definitely use again, Usually get my smeg parts from smeg services but was astounded when they Read more
Product,Price,Delivery. Excellent. Recommended. Read more
Jon Dewing
with paypal guest checkout - quick and simple followed up by good delivery Read more
Richard paskin
Items arrived on time and fit well into hood Read more
Neil Howells*


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Poppie Jones Tiered Ruffle Sequined Tote
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Looks like you've got more shoe shopping to do - have fun!
Item # 233991
Poppie Jones brings us this fun and flirty tote with ruffles and sequins. With this hot look and plenty of space, you can take this bag to work, school, or just our shopping.
- Fabric tote with tiered ruffles and sequins
- Top snap closure
- Flat straps with 11" drop
- Fabric lining with zip pocket and two slip pockets
- Dimensions: 15" x 3" x 15"
- Imported
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September of 1989 began an unprecedented series of natural disasters when Hurricane Hugo hit South Carolina. Little did we know at the time, this would begin a pattern of at least two major events per year for the next 10 years that would change the way insurance companies treated both consumers and their employees.
These events would include at least one major hail storm per year coupled with many others scattered throughout the southwest including hurricanes and earthquakes. Hurricane Andrew hit in August of 1992, becoming the costliest disaster on record for insurance companies with billions of dollars paid out in claims.
As a result, the "loyalty" from the insurance company once coveted by consumers took a direct turn to a total focus on the bottom line, profitability. No longer did it matter you had been insured with one company for 25 years with no claims, once you had a string of bad luck, you were canceled. (more…)


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Miu Miu Suede Bow Flat
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Looks like you've got more shoe shopping to do - have fun!
Item # dsw12prod4180281
With a collection of beautifully crafted footwear, Miu Miu evokes a luxurious sense of freedom and intimacy with attention to detail and high quality. This rhinestone-encrusted suede flat makes it the perfect addition to your shoe collection.
- Suede upper
- Decorative bow
- Cut-away lip detail
- Round toe
- Fully lined in leather
- ½" rhinestone-encrusted heel
- Leather sole
- Made in Italy
- View more Women's Miu Miu Shoes
- Online Exclusive!
By: cocoKo
Location: Korea, South
August 18, 2013
It's so cute!
"shape and meterial are good.Most of all, heel isgorgeous!I choose black color but I wanna buy another color :DBut size is slightly longer than other brand."
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