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Immunotherapy: Facts and Hopes
Cancer Therapy: Preclinical
Smac Modulates Chemosensitivity in Head and Neck Cancer Cells through the Mitochondrial Apoptotic Pathway
Quanhong Sun, Xingnan Zheng, Lin Zhang and Jian Yu
Quanhong Sun
Xingnan Zheng
Lin Zhang
DOI: 10.1158/1078-0432.CCR-10-2262 Published April 2011
Purpose: Overexpression of inhibitors of apoptosis proteins (IAP) contributes to therapeutic resistance. Second mitochondria-derived activator of caspase (Smac) promotes caspase activation by binding to IAPs upon release from the mitochondria. IAP antagonists, also called SMAC mimetics, are promising anticancer agents modeled after this mechanism. We investigated the role and mechanisms of Smac- and Smac mimetic–mediated chemosensitization in head and neck squamous cell carcinoma (HNSCC) cells.
Experimental Design: The effects of SMAC knockdown, SMAC overexpression, and a small molecule Smac mimetic on the chemosensitivities of HNSCC cells were determined. The mechanisms of Smac- and Smac mimetic–mediated chemosensitization were investigated by analyzing growth suppression, the mitochondrial apoptotic pathway, caspase activation, and IAP proteins. The therapeutic responses of HNSCC cells with different levels of Smac were compared in xenograft models.
Results:We found that Smac mediates apoptosis induced by several classes of therapeutic agents through the mitochondrial pathway. SMAC knockdown led to impaired caspase activation, mitochondrial membrane depolarization, and release of cytochrome c. A small molecule Smac mimetic, at nanomolar concentrations, significantly sensitized HNSCC cells to gemcitabine-induced apoptosis and restored gemcitabine sensitivity in SMAC knockdown cells, through caspase activation, X-linked IAP dissociation, and mitochondria-associated events, but not the TNF-α pathway. Furthermore, Smac levels modulated the therapeutic response of HNSCC cells to gemcitabine in xenograft models.
Conclusions: Our results establish a critical role of Smac in mediating therapeutic responses of HNSCC cells and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC. Clin Cancer Res; 17(8); 2361–72. ©2011 AACR.
This article is featured in Highlights of This Issue, p. 2069
Translational Relevance
Patients with head and neck squamous cell carcinoma (HNSCC) are often diagnosed with advanced diseases that respond poorly to chemotherapy and radiation therapy. The overall survival of HNSCC patients has not been significantly improved over the past 2 decades. Overexpression of inhibitors of apoptosis proteins (IAP) was reported to be associated with worse prognosis in HNSCC. In this study, we establish a critical role of second mitochondria-derived activator of caspase (Smac), an endogenous inhibitor of IAPs, in mediating therapeutic and apoptotic responses of HNSCC cells in cell culture and xenograft models. Our mechanistic studies revealed that Smac or a small molecule Smac mimetic activates caspases and amplifies apoptotic signaling through mitochondrial damage, but not the TNF-α signaling, in HNSCC cells. Our studies suggest IAPs as useful therapeutic targets and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC through enhanced induction of apoptosis.
Patients with head and neck squamous cell carcinoma (HNSCC) are often diagnosed with advanced diseases that respond poorly to chemotherapy and radiation therapy. As a result, the overall survival of HNSCC patients has not been significantly improved over the past 2 decades (1). Gemcitabine is a chemotherapeutic agent commonly used to treat HNSCC, and often in combination with other modalities such as surgery, radiation, or additional chemotherapeutic agents including cisplatin. The chemo- and radiosensitization properties of gemcitabine are associated with severe mucositis in the majority of patients (2–4). Therefore, novel strategies are needed to improve efficacy and reduce side effects in HNSCC treatment.
Deregulation of programmed cell death (apoptosis) is a major cause of therapeutic resistance (5–7). Apoptosis is blocked through a variety of mechanisms in HNSCC cells. The tumor suppressor p53 is frequently mutated or targeted for degradation by human papillomavirus (HPV) oncoproteins (8), which prevents the induction of proapoptotic BH3-only proteins, such as PUMA and Noxa, in response to DNA damage (9). Overexpression of antiapoptotic Bcl-2 family members (10), such as Bcl-xL and Bcl-2, is also common in HNSCC (11). Widespread overactivation of growth factor pathways, such as EGFR (epidermal growth factor receptor) and STATs, can suppress apoptosis by affecting the expression of several Bcl-2 family members (12). Overexpression of inhibitor of apoptosis proteins (IAP), such as c-IAP2 and X-linked IAP (XIAP), has been reported in HNSCC (13, 14) and other malignancies (15, 16).
Second mitochondria-derived activator of caspase (Smac) is an endogenous inhibitor of IAPs (17). Upon release into the cytosol, Smac binds to IAPs through its N-terminal AVPI domain and relieves the inhibition of caspases by IAPs. However, induction of apoptosis in response to various anticancer agents is not affected by Smac deficiency in murine models (18). Studies using human colon cancer cells revealed a role of Smac in mediating apoptosis to selective classes of agents, such as nonsteroidal anti-inflammatory drugs (NSAID) and TNF-related apoptosis inducing ligand (TRAIL; refs. 17, 19). Several small molecules mimicking the AVPI domain of Smac, also called Smac mimetics, have been developed and showed antitumor effects in combination with other conventional chemotherapeutic agents in preclinical models (20–22). In some cancer cells, Smac mimetics alone promote apoptosis by engaging TNF-α signaling through IAP degradation (23–26). However, the role of Smac in mediating therapeutic responses and the mechanism of Smac mimetic–induced sensitization of cancer cells remain to be defined.
In this study, we investigated the role of Smac in mediating chemosensitivity of HNSCC cells in vitro and in vivo. Our results suggest that Smac regulates the sensitivity of HNSCC to several classes of anticancer agents and modulates gemcitabine-induced apoptosis through the intrinsic/mitochondrial pathway. Smac overexpression, or Smac mimetic compounds, effectively enhanced apoptosis induced by gemcitabine in HNSCC cells through the mitochondrial pathway but not the alternative IAP/TNF-α autocrine or paracrine pathway.
Cell culture and drug treatment
Head and neck cancer cell lines were obtained from the University of Pittsburgh Cancer Institute (UPCI) Head and Neck Cancer program. All cell lines were maintained at 37°C in 5% CO2. Cell culture media included DMEM (Dulbecco's modified Eagle's media; Mediatech) for 1483 cells and RPMI 1640 (Cellgro) for JHU cells and were supplemented with 10% FBS (HyClone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Gemcitabine was purchased from Eli Lilly and Company. Other anticancer agents used in the study include cisplatin, 5-fluorouracil (5-FU), TRAIL (PeproTech), and human TNF-α and its neutralizing antibody (R&D system). TetraLogic Pharmaceuticals supplied the Smac mimetic, GT-A, and control compound GT-C (19). Stock solutions of all small compounds were prepared in DMSO (dimethyl sulfoxide). For treatment with adenovirus, Cells were infected with Ad-PUMA or the BH3-deleted form (Ad-ΔBH3) (27) (multiplicity of infection = 40) for 48 hours. The Noxa expression vector was constructed in pcDNA3.1, using a PCR-mediated method, and confirmed by sequencing and Western blotting.
Antibodies used for Western blotting included those against caspase-8, caspase-3, Myc (Cell Signaling Technology), cytochrome c, α-tubulin (BD Biosciences), caspase-9 (Stressgen Bioreagents), cytochrome oxidase subunit IV (Cox IV; Invitrogen), Bcl-2 (Dako), PUMA (27), p53 (DO1), cIAP-1, cIAP-2 (R&D system), XIAP (Invitrogen), Survivin (Cell Signaling), Bim, Noxa, and Smac (EMD Biosciences). Western blotting analysis was carried out as previously described (28).
Immunoprecipitation
Cells were harvested after 24 hours with or without Smac mimetic treatment (150 nmol/L) in T-75 flasks and resuspended in 1 mL of EBC buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.5% Nonidet P-40) supplemented with protease inhibitors cocktail (Roche Applied Sciences). The cells were disrupted by sonication and then spun at 10,000× g for 10 minutes to collect the cell lysate. For immunoprecipitation (IP), 2 μg of antibodies or control IgG were added to protein G Dynabeads (Invitrogen) for 1 hour followed by incubation with 400 μL cell lysates according to manufacturer's instructions. After the final wash, the beads were mixed with 50 μL of 1× Laemmli sample buffer, heated at 95 °C for 10 minutes, and analyzed by Western blotting.
Adherent and floating cells were harvested, stained with Hoechst 33258 (Invitrogen), and analyzed for apoptosis by nuclear staining assay and flow cytometry (28). For detection of mitochondrial membrane potential change, harvested cells were stained by Mito Tracker Red CMXRos (Molecular Probes) for 15 minutes at 37°C and analyzed by flow cytometry using the FL3 channel, according to the manufacturer's instructions. For colony formation assays, equal numbers of cells were subjected to various treatments and plated into 12-well plates at different dilutions. Colonies were visualized by crystal violet staining 11 to 14 days after plating as previously described (28). Each experiment was carried out in triplicate and repeated at least twice.
Analysis of cytochrome c release
Mitochondrial and cytosolic fractions were isolated from treated cells by differential centrifugation as previously described (27, 29). Concentrations of cytosolic fractions obtained from different samples were normalized using a protein assay dye reagent from Bio-Rad. All fractions were mixed with equal volumes of 2× Laemmli sample buffer and subjected to Western blotting analysis.
Stable SMAC knockdown and SMAC-overexpressing cells
SMAC short hairpin RNA (shRNA) was constructed using the pSUPER vector (Oligoengine) as described (30). Puromycin-resistant clones were isolated as previously described (31). Western blotting was used to identify stable clones with significant downregulation of Smac in HNSCC lines JHU-012, JHU-019, JHU-022, and 1483. For Smac-overexpressing (SO) cells, JHU-012 and 1483 cells were transfected with an expression construct encoding either Myc-tagged wild-type Smac (AVPI) or mutant Smac with deletion of alanine in the AVPI domain (ΔA; ref. 32), and were selected by G418 (1 mg/mL for JHU-012; 1.2 mg/mL for 1483). Stable clones expressing Smac were identified by Western blotting. Drug-resistant transfectants without KD or SO behaved similarly to the parental cells in response to chemodrugs tested. The parental (P) cells were therefore chosen as the controls.
Xenograft tumors
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. JHU-012 and 1483 xenografts were established and measured as described (28). In brief, 5- to 6-week-old female athymic nude mice (Harlan) were inoculated with JHU-012 or 1483 (5 × 106 cells/site) on both flanks. Tumors were allowed to establish for 10 days. The tumor volumes were measured in 2 dimensions using a vernier caliper. Mice were randomized into groups (7 mice/group), such that the average tumor volume across the groups was the same. Gemcitabine or vehicle (ddH2O) treatments were administered intraperitoneally at 80 mg/kg thrice on days 10, 13, and 16 (33). For all in vivo experiments, tumor volumes were measured every other day in 2 dimensions and volumes were determined in mm3 using the formula: l × b2 × 0.52 (where l is the larger diameter and b is the smaller diameter of the tumor). Mice were injected i.p. 2 hours before sacrifice with a single dose of bromodeoxyuridine (BrdU) at 150 mg/kg to label cells in S phase. BrdU was dissolved in PBS to a final concentration of 30 mg/mL. Histologic and immunofluorescence analysis for apoptosis and proliferation were carried out on 5-μm frozen sections as described (28).
Statistical analysis was carried out using GraphPad Prism IV software. All P values were calculated by the Student's t test, and P < 0.05 was considered significant. Means ± 1 SD were displayed in figures where applicable.
Smac mediates apoptosis induced by therapeutic agents in HNSCC cells
To determine a potential role of Smac in chemotherapeutic agent–induced apoptosis in HNSCC cells, we first analyzed several biochemical markers of apoptosis following gemcitabine treatment. Gemcitabine was found to induce cytosolic release of cytochrome c and Smac and caspase 3 activation in 4 HNSCC lines including JHU-012, 1483, JHU-019, and JHU-022 cells (Fig. 1A and data not shown). We then generated stable SMAC knockdown (KD) cells in these 4 lines by shRNA-mediated gene silencing. Two independent SMAC-KD clones of each line were produced. SMAC-KD cells were found to be resistant to gemcitabine-induced apoptosis, compared with parental cells (Fig. 1B and Supplementary Fig. S1). SMAC-KD partially rescued long-term cell growth suppression induced by gemcitabine in JHU-012 and 1483 cells (Fig. 1C). In addition, SMAC-KD significantly blocked apoptosis induced by other therapeutic agents, including cisplatin, 5-FU, and TRAIL in HNSCC cells (Fig. 1D). These data show that Smac mediates apoptosis induced by several classes of anticancer agents in HNSCC cells.
Smac mediates apoptosis induced by therapeutic agents in HNSCC cells. A, gemcitabine (Gem) induced release of Smac and cytochrome c and activation of caspase-3. JHU-012 and 1483 cells were treated with 50 μmol/L gemcitabine for 48 hours. Smac and cytochrome c (cyto c) in the cytosolic and mitochondrial fractions were analyzed by Western blotting. Tubulin and CoxIV were used as controls for fraction and loading. B, SMAC-KD blocked gemcitabine-induced apoptosis. Top, examples of stable knockdown of SMAC clones in indicated HNSCC lines were identified by Western blotting. Bottom, apoptosis was analyzed by nuclear fragmentation assay. P, parental cells; KD1 and KD2, 2 independent knockdown clones. C, SMAC-KD enhanced clonogenic survival of HNSCC cells following gemcitabine treatment. JHU-012 and 1483 cells were treated by 10 μmol/L gemcitabine or left untreated (un) for 6 hours, then plated at 1:500 dilution (∼400 cells/well) in 12-well plates and allowed to form colonies for 14 days. Top, representative pictures of the colonies. Bottom, the colonies containing more than 50 cells were enumerated and relative survival calculated with untreated cells set at 100%. D, SMAC-KD blocked apoptosis induced by multiple therapeutic agents. JHU-012 and 1483 cells were treated with indicated agents for 48 hours. Apoptosis was measured by nuclear fragmentation assay. Cis, cisplatin (50 μmol/L), 5-FU (50 μg/mL), and TRAIL (100 ng/mL). **, P < 0.01; *, P < 0.05, KD versus P.
Smac mediates gemcitabine-induced apoptosis through the mitochondrial pathway
We further examined the potential mechanism of Smac-mediated and gemcitabine-induced apoptosis. Overexpression of Bcl-2 blocked apoptosis induced by gemcitabine in both JHU-012 and 1483 cells (Fig. 2A), suggesting an important role of the mitochondrial pathway. We then compared biochemical markers of the mitochondrial pathway in parental and SMAC-KD cells following gemcitabine treatment. Consistent with reduced apoptosis, release of cytochrome c and activation of caspases-3, -8, and -9 were significantly attenuated in SMAC-KD cells, compared with parental cells (Fig. 2B and C). In addition, mitochondrial membrane depolarization was significantly blocked in SMAC-KD cells (Fig. 2D).
Smac mediates gemcitabine-induced apoptosis through the mitochondrial pathway. A, Bcl-2 suppressed gemcitabine-induced apoptosis. JHU-012 or 1483 cells were transfected with a Bcl-2 expression construct or empty vector followed by 50 μmol/L gemcitabine treatment for 48 hours, respectively. Top, the expression of Bcl-2 was analyzed by Western blotting. Bottom, apoptosis was analyzed by nuclear fragmentation assay. **, P < 0.01, Bcl-2 versus vector or mock (un) transfected group. B, cytochrome c release in parental and SMAC-KD cells treated as in (A) was analyzed by Western blotting in the cytosolic fractions. Tubulin was used as control for loading. C, activation of caspases-3, -8, and -9 was analyzed by Western blotting in indicated cells treated with 50 μmol/L gemcitabine for 48 hours. D, left, mitochondrial membrane depolarization in JHU-012 parental and SMAC-KD cells was analyzed by flow cytometry 48 hours after 50 μmol/L gemcitabine treatment. Right, quantitation of depolarized cells.
DNA damage is known to activate the expression of several BH3-only proteins and the mitochondrial apoptosis pathway through p53 stabilization. We found that BH3-only proteins PUMA, Bim, and Noxa were strongly induced by gemcitabine, whereas p53 was not consistently induced (Supplementary Fig. S2A). SMAC-KD blocked apoptosis induced by exogenous expression of PUMA or Noxa (Supplementary Fig. S2B). In addition, elevated expression of PUMA or Noxa sensitized HNSCC cells to apoptosis induced by gemcitabine (Supplementary Fig. S2C). These data suggest that regulation of the Bcl-2 family of proteins is likely to be upstream of Smac, and Smac mediates gemcitabine-induced apoptosis and caspase activation by promoting mitochondrial damage, such as membrane depolarization and cytochrome c release, in a positive feedback loop (19, 34, 35).
SMAC overexpression potentiates gemcitabine-induced apoptosis
The N-terminal AVPI residues of cytosolic Smac mediate caspase activation (36). To determine whether this function of Smac is important for apoptosis induced by chemotherapeutics in HNSCC cells, we stably expressed either a wild-type (AVPI) or mutant Smac, containing a deletion of alanine in the AVPI domain (ΔA) that abolishes the interactions between Smac and IAPs (19, 32), in JHU-012 and 1483 cells. Expression of exogenous, mature Smac was lower than that of the endogenous Smac in the stable lines (Fig. 3A, Smac). Nonetheless, gemcitabine-induced apoptosis and caspase activation were potentiated by the expression of the wild-type, but not the mutant Smac (Fig. 3A and B). Furthermore, wild-type, but not mutant Smac, enhanced growth inhibition (Fig. 3C and data not shown) and mitochondrial membrane depolarization induced by gemcitabine (Fig. 3D). These results suggest that elevated levels of Smac sensitize HNSCC cells to anticancer drug-induced apoptosis and growth inhibition by promoting caspase activation and mitochondrial damage.
SMAC overexpression potentiates gemcitabine-induced apoptosis. A, generation of stable wild-type (WT) SMAC-SO cells and mutant (ΔA) cells. P, parental cells; ΔA, mutant SMAC overexpression cells; SO1 and SO2, independent SMAC overexpression clones. Left, Smac expression was analyzed by Western blotting in indicated cell lines. Right, apoptosis in JHU-012 or 1483 cells following indicated treatment for 48 hours was analyzed by nuclear fragmentation assay. **, P < 0.01, SO versus P. Gem, 50 μmol/L and TRAIL, 100 ng/mL. B, the indicated cell lines were treated with gemcitabine (50 μmol/L) for 48 hours. Activation of caspase-3 was analyzed by Western blotting. C, long-term cell growth was assessed by colony formation assay as in Figure 1 C. Cells were treated with 10 μmol/L gemcitabine for 6 hours before plating. Top, representative pictures of colonies. Bottom, quantitation of colony numbers with untreated cells set at 100%. **, P < 0.01, SO versus P. D, mitochondrial membrane depolarization was analyzed by flow cytometry 48 hours after 50 μmol/L gemcitabine treatment in indicated cells.
A Smac mimetic potentiates gemcitabine-induced apoptosis through the mitochondrial pathway
The requirement of the AVPI domain for Smac function prompted us to test whether pharmacologic agents that mimic this domain can enhance gemcitabine-induced apoptosis. An active Smac mimetic compound GT-A at nanomolar concentrations, but not the control compound GT-C, sensitized HNSCC cells to gemcitabine-induced apoptosis (Fig. 4A). GT-A at 100 nmol/L markedly enhanced gemcitabine-induced apoptosis in JHU-012 cells, increasing from 32% to 69% at 48 hours with 50 μmol/L gemcitabine (Fig. 4A), which is associated with markedly enhanced caspase-3 activation and cytochrome c release (Fig. 4A and Supplementary Fig. S3A). Combinations of gemcitabine with GT-A, but not GT-C, inhibited long-term survival and growth of HNSCC cells more effectively compared with gemcitabine alone (Supplementary Fig. S3B and C). The GT-A compound also sensitized HNSCC cells to cisplatin-induced apoptosis and long-term growth suppression (Supplementary Fig. S4). GT-A or the control compound alone up to 1 μmol/L did not have detectable growth inhibitory or apoptotic effects on HNSCC cells, or on caspase activation or cytochrome c release (Fig. 4A and B and data not shown).
The Smac mimetic potentiates gemcitabine-induced apoptosis. Cells were treated with 50 μmol/L gemcitabine with or without 100 nmol/L control (GT-C) or active (GT-A) Smac mimetic compound for 48 hours or as indicated. A, left, apoptosis induction was determined by nuclear staining in 4 HNSCC lines at 48 hours. **, P < 0.01; *, P < 0.05, GT-A + Gem versus GT-C + Gem. Right, caspase-3 activation was analyzed at 48 hours by Western blotting. B, JHU-012 and 1483 cells were incubated with TNF-α Ab (5 μg/mL) for 1 hour and then treated with gemcitabine alone or combined with GT-A for 48 hours. Apoptosis was determined by nuclear fragmentation assay. **, P < 0.01, GT-A + Gem versus GT-C + Gem. C, the indicated cells were treated with 100 nmol/L GT-A. The expression of cIAP-1, cIAP-2, XIAP, and survivin was analyzed by Western blotting at indicated time points. D, the indicated cells were treated with 150 nmol/L GT-A for 24 hours. Left, the whole cell extracts (5% input) were analyzed for the expression of XIAP, survivin, and Smac. Right, the cell extracts were immunoprecipitated (IP) with IgG control or Smac antibody and blotted for XIAP or Survivin.
Smac mimetics were recently reported to induce rapid degradation of cIAP-1/2, leading to NF-κB activation, TNF-α secretion, and apoptosis in some cancer cells (23–26). To probe this potential mechanism in Smac mimetic–induced chemosensitization of HNSCC cells, we treated the cells with TNF-α neutralizing antibody before exposing them to gemcitabine and GT-A. However, the TNF-α antibody did not block apoptosis induced by the gemcitabine and GT-A combination in JHU-012, JHU-019, JHU-022, or 1483 cells (Fig. 4B and data not shown). In contrast, the TNF-α neutralizing antibody effectively blocked apoptosis induced by TNF-α alone or by the TNF-α and GT-A combination in both JHU-012 and JHU-019 cells (Supplementary Fig. S6A), as well as apoptosis induced by GT-A in HT-29 cells as reported before (Supplementary Fig. S6B). We further determined the levels of cIAP-1/2 at several time points following GT-A treatment. The GT-A compound induced a rapid downregulation of cIAP-1/2, but cIAP-2 levels restored within 18 hours, long before significant levels of apoptosis (Fig. 4C). Reduced levels in cIAP-1/2 proteins were not due to decreased mRNA levels (Supplementary Fig. S5).
Other IAP proteins, such as XIAP (36) or Survivin (37, 38), can bind to Smac either in the cytosol or mitochondria to suppress apoptosis. Therefore, the Smac mimetic might promote dissociation of endogenous Smac from XIAP or survivin via competitive binding. The treatment of Smac mimetic did not affect the levels of XIAP, survivin, or Smac but induced a complete dissociation of Smac and XIAP within 24 hours in HNSCC cells (Fig. 4D). The interactions between Smac and survivin were unaffected by GT-A (Fig. 4D). These studies established that Smac mimetic–induced chemosensitization in HNSCC cells is mediated through enhanced caspase activation and mitochondrial damage but not the TNF-α signaling. Nonetheless, the TNF-α signaling is intact and synergizes with the Smac mimetic to induced apoptosis in HNSCC cells.
A Smac mimetic restores gemcitabine sensitivity in the SMAC-KD cells
SMAC-KD cells are resistant to gemcitabine-induced caspase activation and apoptosis compared with parental cells (Figs. 4A and 5A). We expected the GT-A compound to restore these events, bypassing the need for Smac protein. Indeed, the active compound GT-A, but not the control compound GT-C, restored apoptosis induced by gemcitabine in SMAC-KD cells (Fig. 5A). The GT-A compound markedly enhanced caspase activation and cytochrome c release (Fig. 5B and C) and fully restored mitochondrial membrane depolarization in SMAC-KD cells (Fig. 5D). These data suggest that activation of the mitochondrial apoptotic pathway by Smac or Smac mimetics can potentially overcome gemcitabine resistance.
Smac modulates gemcitabine sensitivity of HNSCC in vivo
The Smac mimetic restores gemcitabine sensitivity in SMAC-KD cells. SMAC-KD cells were treated with 50 μmol/L gemcitabine with or without 100 nmol/L control (GT-C) or active (GT-A) Smac mimetic compound for 48 hours. A, apoptosis was analyzed by flow cytometry. B, caspase-3 activation was analyzed by Western blotting. C, cytochrome c release was analyzed by Western blotting. D, mitochondrial membrane depolarization was analyzed by flow cytometry.
To assess whether Smac modulates therapeutic responses in vivo, parental cells, SMAC overexpression (SMAC-SO) or SMAC-KD HNSCC cells were injected subcutaneously into the flanks of BALB/c (nu/nu) nude mice to establish xenografts. Gemcitabine was administered i.p. into tumor-bearing mice on 3 occasions. Comparing with water control, gemcitabine treatment resulted in 70.5% (P < 0.01) and 41.4% (P < 0.01) growth inhibition in 1483 parental and SMAC-KD tumors, respectively (Fig. 6A). Similarly, gemcitabine administration inhibited JHU-012 parental and SMAC-KD tumors by 71.8% (P < 0.01) and 33.8% (P < 0.01), respectively (Supplementary Fig. S4A). Meanwhile, gemcitabine treatment resulted in 21.2% (P < 0.01) and 57.2% (P < 0.01) growth inhibition in parental and SMAC-SO tumors, respectively (Fig. 6B). The differences between the responses of the tumors with different SMAC genotypes were statistically significant, whereas little or no difference was found in the efficiency or growth rate in tumor establishment in the absence of treatment (Figs. 6A and B and Supplementary Fig. S7A).
Smac modulates gemcitabine sensitivity of HNSCC cells in vivo. The responses of SMAC-KD or SO xenograft tumors to gemcitabine were compared with that of 1483 parental (P) xenograft tumors. Gemcitabine (80 mg/kg/d) was administrated to tumor-bearing mice on days 10, 13, and 16 as indicated by arrows. A, growth curves of 1483 parental (P) and SMAC-KD1 xenograft tumors (n = 7/group) subjected to gemcitabine or control treatments. **, P < 0.01. KD1 + Gem versus P + Gem, and KD1 + Gem versus KD1 + ddH2O. B, growth curve of 1483 parental and SO1 tumors (n = 7/group) subjected to gemcitabine or control treatment. *, P < 0.05, SO1 + Gem versus P + Gem; **, P < 0.01, SO1 + Gem versus SO1 + ddH2O. C, frozen sections of indicated 1483 tumors 48 hours after the second injection were analyzed by hematoxylin and eosin (H&E) staining. Apoptosis and proliferation were analyzed by TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) staining (red) and BrdU incorporation (red), respectively. The nuclei were counterstained by 4′,6-diamidino-2-phenylindole (blue). Magnification, 400×. D, index of TUNEL-positive or BrdU-labeled cells in 1483 tumors with indicated genotypes 24 hours after the second gemcitabine injection. **, P < 0.01, KD1 (or SO1) + Gem versus P + Gem.
Analysis of tumor sections after the second gemcitabine injection (day 15) revealed significantly lower apoptosis and higher cell proliferation in SMAC-KD tumors compared with parental tumors. In contrast, SMAC-SO tumors showed more extensive apoptosis (13% vs. 5%) and lower proliferation than parental tumors (Fig. 6D and Supplementary Fig. S7B). These results show that the levels of Smac modulate the therapeutic responses of HNSCC cells to gemcitabine in vivo through apoptosis.
Smac in anticancer agent–induced apoptosis in HNSCC cells
IAP family members are frequently overexpressed in many solid tumors including HNSCC. Overexpression of IAPs was reported to be associated with worse prognosis in HNSCC (13, 14). Biochemical studies indicate that IAP proteins are antagonized by Smac in mammals, which promotes caspase activation and apoptosis through its N-terminal AVPI motif (21). The structural basis of such interactions led to the development of several small molecule Smac mimetics, also called IAP antagonists, which are believed to compete with caspases for IAP binding and consequently release caspases to promote cell death (20–22). Despite extensive biochemical data, SMAC-KO mice or mouse fibroblasts show limited if any alteration in apoptosis (18). Our study showed, for the first time in HNSCC cells, that Smac plays an important role in apoptosis induced by several classes of anticancer agents, and elevated Smac levels or a Smac mimetic compound potentiates therapeutic responses of HNSCC cells by promoting apoptosis.
Our observations are consistent with the notion that a requirement of Smac in apoptosis appears to be cell-type- and agent-dependent (30, 35). Overexpression of Smac or Smac mimetics can potentiate anticancer effects of chemotherapeutic agents and irradiation in glioma, hepatoma, neuroblastoma, glioblastoma, or pancreatic carcinoma cells (25, 39–41). It is of interest to note that the killing or sensitizing effects of Smac or Smac mimetics appear somewhat selective toward cancer cells compared with normal or untransformed cells (16). The precise mechanisms of this differential sensitivity remain unclear, which might be explained partly by the addiction of cancer cells to overexpression of IAPs or perhaps alterations in other upstream regulators such as the Bcl-2 family of proteins and the death receptors (16).
Signaling events in Smac-mediated apoptosis and the mitochondria
Emerging evidence suggests that induction of BH3-only proteins by therapeutic agents might be a universal mechanism underlying favorable and apoptotic responses of cancer cells (28, 31, 42–45). In HNSCC cells, the BH3-only subfamily plays a critical role in regulating their survival, whose expression is suppressed by an oncogenic form of p63 overexpressed in majority of squamous cancers (31, 46, 47). Our data show that BH3-only proteins PUMA, Bim, and Noxa are induced by gemcitabine mostly likely through a p53-independent mechanism, and SMAC-KD blocked apoptosis induced by PUMA or Noxa. Therefore, multiple BH3-only proteins might be involved in promoting mitochondrial damage and Smac release during gemcitabine-induced apoptosis. In addition, SMAC-KD abrogated apoptotic responses to several anticancer agents, which is rescued by the Smac mimetic. Despite a clear role of Smac in activating caspases following its release from the mitochondria, our data suggest that the release of apoptogenic proteins might not be independent, and a complex and positive feedback mechanism might exist to regulate mitochondrial outer membrane permeability (MOMP) and caspase activation (30, 34, 35, 48).
Mechanisms of Smac mimetic–induced chemosensitization in HNSCC cells
Smac mimetics can induce rapid degradation of cIAP-1/2, leading to TNF-α–dependent apoptosis through the NF-κB signaling in some cells (23–26, 49). In this study, the GT-A compound was also found to induce rapid downregulation of cIAP-1/2, with cIAP-2 levels restored long before apoptosis. These observations coupled with failure of the TNF-α neutralizing antibody to block apoptosis induced by the gemcitabine and Smac mimetic combination would suggest that the TNF-α signaling is unlikely to be responsible for the chemosensitization effects of Smac mimetics. Rather, a more direct mechanism engaging caspase activation and mitochondrial damage is mediated, at least in part, via the dissociation of endogenous Smac and XIAP. Because Smac is found predominantly in the mitochondria of cells such as HNSCC cells, it is reasonable to predict that this dissociation occurs at the mitochondria. It is also possible that Smac mimetics activate additional proapoptotic proteins (50) by displacing them from IAPs either in the cytosol or mitochondria. The selective involvement of TNF-α signaling (23–26, 49) or the mechanism described here might reflect the cell-type–specific role of endogenous Smac and/or the structural and functional differences of the small molecule Smac mimetics.
Implications on novel combination therapies in HNSCC
In phase II studies, gemcitabine in combination with other chemotherapeutics or radiation has shown improved response rates in HNSCC patients with advanced diseases, compared with single-agent regimes (2–4). However, severe mucositis is a common complication in combination settings. Our data suggest that this side effect might be reduced without compromising therapeutic efficacies, potentially by using lower doses of gemcitabine with Smac mimetics. Smac mimetics might be useful as sensitizers for other anticancer agents to boost apoptosis in otherwise resistant tumor cells. Several apoptotic blocks exist in cancer cells and the Bcl-2 family of proteins have become promising targets with the development of a class of so called BH3 mimetics, or Bcl-2 antagonists (51, 52). In HNSCC cells, induction of BH3-only proteins in response to 5-FU or cisplatin is often blocked by defective p53 signaling (28), whereas their induction by gemcitabine appears largely p53 independent (this study). Even modest overexpression of BH3-only proteins, such as PUMA or Noxa, can sensitize HNSCC cells to these agents, independent of p53 status. Smac and BH3-only proteins act at different steps of apoptosis and participate in a positive feedback loop to activate caspase and mitochondrial damage. Therefore, targeting either or both steps with Smac and BH3 mimetics depending on the genetic background of the tumors might bring us one step closer to individualized HNSCC treatment.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work is supported by NIH grant CA129829, American Cancer Society grant RGS-10-124-01-CCE, and FAMRI (J. Yu) and by NIH grants CA106348, CA121105 and American Cancer Society grant RSG-07-156-01-CNE (L. Zhang).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The authors thank other members of Yu and Zhang laboratories for helpful discussions.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/.
Received August 25, 2010.
Revision received December 23, 2010.
Accepted January 3, 2011.
©2011 American Association for Cancer Research.
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Clin Cancer Res April 15 2011 (17) (8) 2361-2372; DOI: 10.1158/1078-0432.CCR-10-2262
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Home Sports
Inzaghi: "It's a final, there are no favorites. Glad that Zhang is here"
The Inter coach on the eve of the Super Cup final: "Juve more used to matches like these, but we want to give the fans a joy: the reasons will be very important"
"The finals are all special matches, they can be decided by the single episode. We will have to play a game of racing, aggression and determination. There will be moments in which Juve will put us in difficulty, we will have to be good at getting out of it." Simone Inzaghi is looking for the third victory in the Super Cup against Juventus, after the two on the Lazio bench. But in the press conference on the eve of the San Siro match he doesn't want to hear about a favorite Inter Milan: "I was lucky enough to play so many finals, there aren't any favorites in these matches. We face a healthy team, with eight useful results. consecutive and after an extraordinary comeback. It is a team used to playing finals, we are in an excellent moment and we will play it with our weapons. The episodes and motivations will be very important ".
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Rank and Zhang
Inzaghi returned to the presumed greater pressure given by the first place in the standings: "I have said it several times: the primacy must be a stimulus for us. On August 20, the clear favorite for the Scudetto seems to me to be Juventus, we were between the teams immediately behind. Until the end it will be an open fight: Milan and Napoli play very well, Atalanta have been at this level for years, Juventus are doing well. " To get closer to the match, the presence of Steven Zhang in Milan these days is important: "I'm happy that he is physically there. In all these months I have always felt him very close, we heard often and we texted each other before and after each game. Tomorrow we hope to have a great match for him, for the fans, for the club. We have to prepare ourselves as best we can. " The comparison with the championship match that ended 1-1 is superfluous: "I saw it again these days, it was a very tight match. We were both late, it was a bad match and played underneath by both teams".
See also Inzaghi voiceless, there is deputy Farris: "There is regret for the equal. Dybala? The company will take care of it"
Church and the attack
Juve will not have Cuadrado, De Ligt and Chiesa ("I want to do good luck to Federico: besides being a great Juve player he is a heritage of Italian football"), but Inzaghi does not trust: "Juve it's a team with an important squad, Allegri will know how to replace them in the best possible way and will recover Bonucci and Chiellini, so they will have a competitive team. " A point on the conditions of his boys: "Today we have training, yesterday I saw on the pitch those who had not played since the beginning against Lazio. They are all in good condition, including Dzeko and Correa. In front of me I have all players who are doing well , in every match I will choose the best couple: having them all is a great asset ".
Handanovic
Samir Handanovic intervened in the conference alongside Inzaghi, who was asked about the possible future coexistence with André Onana: "The voices do not bother me: the competition in the role is welcome. The important thing is to have self-confidence, not I think of what will come: I think of today and tomorrow, I live for the day ". The Nerazzurri goalkeeper removes a few pebbles after criticizing the goal against Lazio: "When Inter are unbeaten it's thanks to the defenders, when they concede a goal it's Handanovic's fault. De Vrij misjudged the trajectory, my mistakes and Skriniar's arrived accordingly. Let's try to make as few mistakes as possible and always keep the ball on us. "
See also 5 things you did not know about Ezequiel Centurión, the archer Gallardo called to be a substitute for Armani
January 11, 2022 (change January 11, 2022 | 14:03)
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redpajama_set_name: RedPajamaCommonCrawl


Historical novel, set in the 16th century, with privateer Sir Francis Drake hunting Spanish treasure ships. According to Wikipedia: "George Alfred Henty (8 December 1832 - 16 November 1902), referred to as G. A. Henty, was a prolific English novelist, special correspondent, and Imperialist born in Trumpington, Cambridgeshire, England. He is best known for his historical adventure stories that were popular in the late 19th century. His works include Out on the Pampas (1871), The Young Buglers (1880), With Clive in India (1884) and Wulf the Saxon (1895)."
Chapter 3: On the Spanish Main.
Chapter 4: An Unsuccessful Attack.
Chapter 6: In the Woods.
Chapter 7: An Attack in Force.
Chapter 8: The Forest Fastness.
Chapter 11: The Marvel of Fire.
Chapter 12: Across a Continent.
Chapter 13: Through the Cordilleras.
Chapter 14: On the Pacific Coast.
Chapter 15: The Prison of the Inquisition.
Chapter 17: The Golden Hind.
Chapter 18: San Francisco Bay.
The lad of whom they were speaking was a bright-faced boy, of some fifteen years of age. He was squarely built, and his dress differed a little from that of the fisher lads standing on the beach. Who is he? asked Captain Drake.
It would have been no easy matter to get on board the ship; so, having done his work, the lad turned to make his way back to the shore. A thick rope was fastened, at once, by those of the crew who still remained on the deck of the vessel, to the lighter one; and those on shore began to pull it rapidly in; but, ere the knotted joint reached the shore, a cry from all gathered on the beach showed that the brave attempt of the Otter had been useless. A tremendous sea had struck the ship, and in a moment it broke up; and a number of floating fragments, alone, showed where a fine vessel had, a few minutes before, floated on the sea.


text:
What is LARC and how can it tackle Pakistan's runaway population problem?
A lack of family planning and knowledge about contraceptive methods is leading to a surge in Pakistan's population.
Zofeen T. EbrahimPublished 11 Jul, 2017 07:30am
Situation in Sindh
All cleaned up, his tiny head visible from under the colourful beret, the rest of the body wrapped tightly in a cloth, the new born was gently passed on to an elderly man standing outside in the passage of the labour room at KMC's Gizri Maternity Hospital. The mood is more solemn than of jubilation. The man leaned over and began reciting the Azaan loudly, first in one ear and then the other. The little one, his eyes wide open, listened to it intently. Five minutes later, another baby was brought out and handed over to the man and the ceremony was repeated.
Oblivious to that, just a few feet away, Shakila Ramzan, a reed of a woman shouted out 'Rabia ke saath' and minutes later a nervous-looking young man appeared, an older woman trailing behind him. The next half hour was spent by Ramzan trying to convince Muaqaddas Ali, 24, soon to become a father second time around, the advantages of family planning, and that too, soon after delivery.
With his wife Rabia soon to deliver, Ramzan had a small window of opportunity to get Ali's consent to allow the hospital administration to insert a T-shaped Intrauterine Contraceptive Device (IUCD) into his wife's uterus while she was still on the delivery table.
The couple didn't want any more children, but Ali seemed reluctant to have an IUCD inserted in her wife. They had refused during antenatal clinics which his 18-year old wife had been visiting regularly. "After our first child, we were told by the doctors at the previous hospital that this FP method was not suitable and may cause a lot of problems," Ali explained of his reluctance. He said they would decide after 40 days.
"It's really difficult to convince husbands to give consent and sometimes we have to do it multiple times. It can be quite tiresome" - Maria Irfan
Gentle cajoling did the trick and Ali signed the consent form. "I have to talk so much every day, it's quite draining," said Ramzan, as she wiped the sweat off her face and straightened the dupatta over her head. She also said that if the long acting IUCD was not inserted immediately after delivery, women would have to wait six weeks. "During that period, she may get pregnant!"
In another part of town, in another hospital, the situation is similar. Maria Irfan, staff incharge at the government-run Sindh Empoyees Social Security Institution hospital in Landhi, is a trained counselor for LARC (long acting reversible contraceptive). "It's really difficult to convince husbands to give consent and sometimes we have to do it multiple times. It can be quite tiresome," she echoes Ramzan's woes.
Both feel, lack of education is a big curse. "If only these women were even slightly educated, our tasks would be made so much easier," said Ramzan.
Myths come in the way of contraception
Close by, waiting patiently to speak to Ramzan, was Rubina Sachal a mother of seven and having gone through 11 abortions. "Please remove the coil as I bleed heavily and suffer from severe pain in my back; and I'm gaining a lot of weight as well," requests an adamant Sachal. The women in the neighbourhood had scared her that the IUCD may have travelled up into her intestines.
Ramzan sat her down, addressed her complaints and convinced her to come back after a month while writing down a prescription for an iron supplement. "Take these tablets regularly and if you still suffer from these symptoms we will remove it," she told her gently with a reassuring smile, allaying the woman's fears.
Ramzan revealed that the woman had visited her twice before and she had been able to convince her not to get the contraception device removed. "If she does, she will get pregnant as her husband is idle and she won't take any precaution," she said knowingly.
"These women come with the weirdest misconceptions; and these would be hilarious if it were not so tragic. Even a little spotting will be exaggerated and considered bleeding," she said. Clearly the factors that she has had 11 abortions, seven births and that she is the only bread earner (the husband does not work) in the house are disregarded for her having health issues.
The PDHS (Pakistan Demographic and Health Survey) 2012–2013 states that twice as many women (28.3pc) from poorer households complain of side effects from Family Planning methods than wealthier women (14pc). The findings reflect poor quality of services (weak counseling about side effects and choice of method) at facilities serving the poor.
Ramzan said if something goes wrong with even one woman the method receives a bad reputation and its consequences spread like wildfire, not just in that neighbourhood but adjacent ones as well. By the same token the satisfaction and acceptance does not seem to spread as fast.
The exploding population
Pakistan just had its highly anticipated census and while the nation awaits its results, demographer Dr. Farid Midhet, heading the John Hopkins University affiliated international organisation Jhpiego in Pakistan, is certain there won't be too many surprises from results projected by various international organisations.
Right now the estimated population of the country is 195 million according to the United Nations (UN), making it the sixth most populous country in the world. By 2050, according to Population Reference Bureau, if things remain the same, Pakistan would remain in sixth position exceeding, 300 million people given its population growth rate.
Pakistan has one of the highest fertility rates in the region apart from Afghanistan and the lowest rate of contraceptive use (35pc) despite women showing an aversion to further procreation.
Dr. Zeba Sathar, Pakistan country director of the Population Council told Dawn.com that seven million women either don't want to bear more children or want to space births, but are unable to do so.
"They are unable to do so because of the lack of services or choice of contraceptives near or where they live," she explains, adding, "They also need more information and care regarding side effects to curb high discontinuation rates."
Sathar believes, "more choices of providers near their homes or community-based workers giving more than a few methods through task sharing, like lady health workers (LHWs) giving injectables in addition to pills and condoms would enhance choices instantly."
Long-acting reversible contraceptives (LARC) are methods of birth control that provide effective contraception for an extended period without requiring user action. They include injections, intrauterine devices (IUDs) and subdermal contraceptive implants.
However, there are doctors like Azra Ahsan, a gynecologist and obstetrician with over two decades of dealing with patients from all strata of society, who believe Pakistan needs a more robust and sure shot way of tackling the runaway population.
They call it LARC - long acting reversible contraceptive, the same that Ramzan is promoting at the hospital in Gizri. There are two such methods - implants and IUCD, although Sathar also includes injectibles (which last for three months).
Although both IUCDs and implants have been around for a long time, even in the west they are not very popular because of past design flaws, difficult insertion and removal demands, or by the side effect and the bad reputation has stayed.
However, the new LARCs have come a long way according to Ahsan.
LARC 20 times more effective than the pill
"The currently available LARC methods are easy to use, safe, long lasting and quickly reversible. In fact, they have fewer contraindications for use," she said, adding, "I'm all for it."
Ahsan, also the technical consultant at the Ministry of Health's National Committee for Maternal & Neonatal Health (NCMNH) clearly favoured inserting IUCDs right after delivery leaving nothing to chance before the mother left the hospital. She says this post-labour procedure, which is done as soon as 10 minutes after delivery, is not difficult or risky, just different. "But imagine the woman can become stress free for as long as 12 years in the case of an IUCD and five years for an implant!"
Concurring with Ahsan, Dr. Midhet added, "LARC can help lower the rate of unintended (both mistimed and unwanted births); it gives the mother time to recuperate after a birth and provides an opportunity to the newborn to be breastfed, thereby prevent stunting and malnourishment." In Sindh, 48pc of children under five suffer from stunting and 15pc from wasting.
Senior technical advisor in Pakistan for Pathfinder, Dr. Tauseef Ahmed, also finds that LARCs provide the "best option" in terms of impact and also in terms of client satisfaction.
In Pakistan, he said, tubal ligation (more commonly known as female sterilisation) and condoms which are more popular, have not proven to have any impact on fertility. "Tubal ligation is preferred by women who have already produced 6-7 children, while condoms [are] associated with inefficacy."
Pathfinder is working with all provincial governments to create costed implementation plan— a five-year clear roadmap for achieving their goals for contraception.
With more and more women delivering at hospitals (the latest Pakistan Demographic and Health Survey of 2012-13 says it's 48pc) it perhaps gives healthcare providers that perfect but small window of opportunity to promote this method.
The reason for the urgency to start women on some form of contraception has been felt for some time. In the past doctors usually waited to discuss an IUCD or implant until a woman's first postnatal checkup, six weeks after delivery. But, Ahsan conveys that most women never show up for this exam.
A recent study conducted in two hospitals in Karachi and Dadu, where over 3,000 women were interviewed, tried to find the reasons behind the high rate (38.2) of unintended pregnancies amongst women attending antenatal clinics in Pakistan and the factors related to FP.
The study found that 89.9pc women had knowledge about at least one of the contraceptive methods, but only 33.4pc reported using them with the most common being condoms (19pc) followed by injectables (9.7pc), the pill (9.6pc), intra uterine device (2.9pc), and implants (2.5pc) among modern methods. Among traditional methods only 14.5pc and 34.5pc of women had knowledge about the rhythm and withdrawal methods, while 13.8pc and 46.1pc of women reported using the rhythm method and withdrawal method respectively.
Unintended pregnancies and poverty
Unintended pregnancy in Pakistan, pointed the study, was higher among women who were younger, poorer, illiterate and living in a rural setting. Often the interval between pregnancies was less than 12 months, and they often had previous history of miscarriage or abortion.
"It makes sense since younger women have higher fertility, higher frequency of sexual intercourse, lower knowledge of contraceptive methods and higher rates of contraceptive failure relative to older women," said Dr Atif Habib, one of the authors of the study, speaking to Dawn.com.
A Population Council report noted that in 2012, there were approximately nine million pregnancies in Pakistan, of which 4.2 million were unintended.
Most unintended pregnancies result in abortions. Abortion is illegal in Pakistan and only permissible to save a woman's life or to provide "necessary treatment" early in pregnancy. Because the law is unclear and healthcare providers have their own personal beliefs, safe abortion services are difficult to obtain and most women who want it carried out resort to clandestine, and unsafe procedures.
A Population Council report noted that in 2012, there were approximately nine million pregnancies in Pakistan, of which 4.2 million were unintended. Of these unintended pregnancies, 54pc resulted in induced abortions and 34pc in unplanned births.
For this very reason, states Dr. Habib's study, over a 5-year period, even if 4pc women switch from current oral contraceptive use to LARC, 25,000 unintended pregnancies and their related abortions and unplanned births can be averted.
Ramzan sees almost two to three cases of post abortion complications daily and she convinces them to get the post partum IUCD inserted to save them from another abortion.
Same is the case at the Landhi hospital. Altaf expresses that while couples think nothing of getting several abortions done to terminate pregnancies, when it comes to using FP they say it's a sin since children are God's gift to them.
In her hospital, unlike in Gizri, implants are more popular. Just in May, she was able to convince 60 women to use this method while only six women were ready for PPIUCD.
Almost five years have passed since LARC has been promoted widely by various organisations and the acceptance rate, is growing. Dr. Laila Shah, director of the Sukh Initiative at Jhpiego is also promoting LARC in 42 public health facility hospitals in four towns of Karachi.
"It's a relatively a new concept in Pakistan, but due to the convenience it's gaining traction, since women don't have to return for FP service. In addition, fewer side effects are experienced in long term protection, she said. In the four towns they are working in, 30pc of delivered women opted for either implants or IUCD.
"I think we need to give credit to the counselors, especially those in labour rooms; they have indeed played a very important role and if we need to make further progress, this brigade of women are key."
For encouraging women to opt for LARC, Shah said dedicated counselors are deputed at selected mother and child health facilities in antenatal clinics and labour rooms.
At the same time, "we need more counselors," said Dr Ahsan and not just in the day time. "Many deliveries take place in the night when there is no one to counsel the woman and her family. Since FP comes under the Population Welfare Department (PWD) and deliveries happen in hospitals, which comes under health department, there remains a huge gap.
Another problem is that till the NGOs oversee counseling and insertion of LARC at government hospitals, the number of women consenting to LARC keeps growing. "But the moment we try to wean ourselves away, the numbers begin to fall drastically," observed Ahsan. She lamented that LARC had not been institutionalised in the manner that it should.
But Shah has a solution. She said the Family Welfare Centres (FWC) run by the PWD and the Reproductive Health Centres (also working under PWD) within the tertiary hospitals can easily be trained and accommodated in wards, since fewer couples visit these centres. In any case they close by 2pm. In Sindh alone, there are about 1,000 FWCs, but only 40pc are functional informed Midhet.
The economics of the situation also poses a problem. The upfront cost is far more expensive than the pills or condoms. But, Shah says, "the cost is covered by the government at their hospitals." "In any case," she went on to reassure, "compared to other methods, LARC is more cost-effective in the long-term."
However, Dr Ahmed of Pathfinder feels LARC has its limitations as not everyone can insert or remove these. "It needs technical competence as well as quality counseling and follow up for managing and treatment of side effects."
For LARC to be successful and have an impact governments hospitals, doctors, midwives as well as counselors all need to go that extra mile. That means training doctors, availability of counseling 24/7 and reassuring patients that if they ever have misgivings, they can come and have the device removed.
To read more about the situation in Sindh and frequently asked questions click on the tabs below.
In 2012, Pakistan along with 68 of the poorest countries made a global commitment to increase contraceptive use among women in those countries at a summit held in London, called the FP2020 . Compared to 2012, there are now 30.2 million additional users of modern methods, which is 25% higher. And though significant, it is still 19.2 million users short of the pace needed to reach the goal of 120 million additional users by 2020.
As a result of the summit, Pakistan made several promises aimed at increasing access to and use of family planning to constrain the growth of its population. These are referred to as Pakistan's FP2020 commitments. Because health is a provincial subject after the 18th amendment in 2010, provincial goals had to be set.
The 2017 London Summit on Family Planning will be held in London, United Kingdom on July 11, under the shadow of the "global gag rule" imposed by the US that prohibits US organisations from assisting aid to foreign organizations that work on abortion counseling, referrals or services which would mean a cut the equivalent of about $600 million in family planning aid.
With all the provinces having their own population policies, Sindh government is the only one that has come up with an approved operational tool to breathe life into the policy. Termed Costed Implementation Plan (CIP), it is worth Rs141bn for five years.
According to Dr Ahmed, in Sindh, the number of implants served during 2015 and 2016 and now 2017 has reached 60,000. "Credit for this goes to Dr Azra Pechuho," he said, adding: "...and the coordination and zeal shown by the Pakistan Primary Health Initiative (PPHI) for pursuing implants after training all the doctors at all basic health units across the province."
In Sindh, there are currently 13 million women of reproductive age, and this number is estimated to reach 15 million by 2020. The total fertility rate in Sindh has declined from 5.1 births in 1990–1991 to 3.9 births in 2012–2013, while the CPR has been stagnant at 29.5pc, with 24pc of women using modern methods of contraception for over a decade. The disparities in the province are visible with 42.7pc CPR in urban areas and 17.4pc in rural areas, while unmet need remains at 21pc.
Sindh's population is projected to increase to 50 million by 2020. There are 1.747 million married women of reproductive age (MWRA) who are the current users of contraception. The users of modern methods of contraception are projected to rise to approximately 3 million by 2020. Therefore, the province of Sindh should expect 1.298 million additional users of modern methods.
Both the public and private sectors are contributing towards provision of contraceptive services. An estimated 45pc of contraceptives are provided by the public sector. The two major sources within public sector are the Population Welfare Department (PWD) and the Department of Health (DOH). The private sector nongovernmental organizations (NGOs) provide 20.5pc of contraceptives; private pharmacies and chemists provide 25.3pc; private doctors contribute 2.1pc; while shops provide 6.5pc of the contraceptives.
Thus, the combined share of private sector is estimated to be 54.4pc. The PWD provides the services through its static and outreach facilities. Likewise, the DOH and People's Primary Healthcare Initiative (PPHI) provide FP services through the static setup and through the massive network of community health workers—the lady health workers (LHW).
To increase access and uptake of FP in Sindh, there are quite a number of areas of concern that need to be addressed. Through a consultative process with key stakeholders, PWD and DOH identified broad priorities to develop six strategic areas for investment in FP. The CIP presents the strategic areas and the rationale for their selection, along with associated costs. The strategic areas are:
Functional Integration: Enhancing strategic coordination and oversight between the population and health sectors at the provincial, district and subdistrict levels regarding functional integration of services at the subdistrict level.
Quality of Care: Ensuring quality of services by enforcing standards, improving providers' skills and ensuring client satisfaction.
Supply Chain Management: Improving contraceptive security to the last mile, including distribution and availability of contraceptives at service delivery points.
Expansion of services: Expanding services with supply- and demand-side interventions for enhancing access, especially to urban slums, peri-urban and rural areas, and creating space and linkages for public-private partnerships to reach vulnerable segments of the population including the poor and youth.
Knowledge and Meeting Demand: Increasing knowledge and meeting the demand for FP services by focusing on MWRA, emphasizing male engagement and young people.
Governance, Monitoring and Evaluation: Strengthening the health and population systems by streamlining policy planning, governance and stewardship mechanisms, and performance monitoring and accountability.
These strategic areas are guided by three principles (1) strengthening existing systems and services; (2) accelerating interventions; and (3) innovating proving ideas, bearing high-impact practices and modern technologies.
In order to achieve the following three objectives:
Enhance CPR from 30pc in 2015 to 45pc by 2020.
Reduce unmet need for FP from 21pc in 2015 to 14pc by 2020.
Ensure contraceptive commodity security up to 80pc for all public sector outlets by 2018.
Implementing the CIP over the next five years will have a significant impact on health, demographic and economic activities. As a result of determined action on the proposed interventions, an estimated 1,848 maternal deaths and 29,470 child deaths will be averted by the year 2020.
Regarding demographic impact, an estimated 1,774,367 unintended pregnancies and 193,332 unsafe abortions will be averted, while PKR 12.187 billion will be saved by implementing the CIP, as an estimated 3,963,060 couple years of protection will be generated.
Punjab's CIP is also ready and is expected to be approved by the end of June or early July, Khyber Pakhutunkhwa's awaits a signal from the chief minister for a briefing (may get approved before the London Summit), and Balochistan should be ready to be presented to the chief minister by August.
To read more about family planning and frequently asked questions click on the tabs below.
Everything you always wanted to know about LARC but were afraid to ask.
Dr. Farid Midhet, who heads Jhpiego in Pakistan, responds to some questions women may have concerning LARC.
Affiliated with Johns Hopkins University, Jhpiego is an international non-profit organisation geared towards providing technical support and training in family planning, and improving maternal and child health in Pakistan for over 15 years.
Currently Jhpiego is taking the lead in institutionalising postpartum family planning across all provinces.
1. I'm in my 40s and our family is complete. I'm thinking of having a coil fitted but have heard scary stuff. Should I go for sterlisation instead?
For you, choices could be, implant and IUCD as both are long term methods and provide protection from 5 to 12 years respectively. While vasectomy and female sterilization are permanent methods and could be a possible option. Mirena is also good for pre-menopausal women.
2. I've also heard of implants. My concern is that it may release certain hormones and then the idea of having some metal inserted under my skin makes me uneasy.
There is no metal in implant, it's a plastic devise with hormone(progesterone) and will not cause any discomfort due to elasticity of its rods. The side effects such as bleeding and spotting may occur in initial few weeks but are clinically insignificant and reduce considerably with time and without any treatment. However, insertion of implants requires service from a trained provider.
3. Between the IUCD and the implants which is more cost effective? Which is more foolproof?
In the Pakistani context, both should be available to women free of cost (or at a highly subsidized cost); therefore, cost is probably not an issue. However, implants are roughly 2-3 times more expensive than an IUCD in terms of manufacturing price. Both are about the same in terms of effectiveness, although hormonal IUCD is slightly more effective. No contraceptive method (except abstinence and removal of the uterus) is 100pc 'foolproof'!
4. I'm in my late 30s and going to have my second child. After that we don't want to have any more children, at least for a while. I've heard of post partum IUCD. Is it safe to have something inserted so soon after delivery? I will be feeding my newborn.
Postpartum family planning IUCD (PPIUCD) and implants (IPPI) are two long term contraceptive that can be given right away after delivery without affecting breast milk. Both methods are extremely safe and effective with fewer side effects. The benefits far outweigh the potential risks and inconvenience. Both methods require a trained health care provider.
5. I'm in my late 30s, and have been told by my friends that I am too old for the pill which I have now been taking for almost eight years?
If you do not smoke, do not have high blood pressure or diabetes and have no genetic history of breast cancer, if you are physically active, not obese, and do not have varicose veins, then you do not need to worry. In general, however, women in late 30s and 40s and on the pill should consult their doctor frequently, primarily to exclude the above risk factors.
6. From among the three - long acting contraceptives - IUCD, implant and injectible, which one is the most reliable?
Implants are probably No. 1, but hormonal IUCD is a close second. I would not include DMPA injection in the long acting method as needs to be repeated every two to three months.
7. How soon can I get pregnant after getting an IUCD or an implant removed?
Right away.
8. I'm a cancer survivor, can I use any of the LARCs?
Yes, if cancer is not hormone sensitive which means not in the reproductive organs then all LARCs can be opted after medical evaluation.
9. Can a woman with diabetes use LARCs?
Yes. Both implant and IUCD can be given to diabetic with special aseptic measures.
A rapid fire round with Dr. Azra Ahsan, consultant obstetrician and gynecologist on IUCD
1. Is it safe?
Yes very safe
2. Does it hurt?
Some discomfort during insertion, none later.
3. Can my partner feel it during sex?
No not at all.
4. Will it interfere with my ability to get pregnant again?
Once removed the fertility is instantly restored
5. How long do I have to wait after giving birth to get an IUCD?
Best if inserted within 10 minutes of childbirth, if not possible then before 48 hours. It can be inserted anytime after 4-6 weeks of childbirth if not inserted within 48 hours.
6. Is it hard to remove?
Removal is a simple procedure, which can be done in a clinic setting.
7. Is there an alternative?
Yes the other possible alternative for a long acting reversible contraceptive is hormonal implant.
To read more about family planning and the situation in Sindh click on the tabs below.
Fatal conception: Stilled life
Family planning may be our last hope
Who's enemy number one?
Ashley caldeira
Europe had implemented all these methods but now Europe has collapsed because they do not have any population. The EOBI system has collapsed because there is no people to support. Family Planning is good but the intention is not to stop children.


text:
Home News Faculty's attention drawn to DACA support, student substance abuse, and bias incident...
Faculty's attention drawn to DACA support, student substance abuse, and bias incident at first meeting of the year
Almost 120 DePauw University faculty members met for the first time this year on Sept. 11 to discuss a plethora of issues. Top issues include support for undocumented students, high risk drinking, and the Humbert Hall bias incident.
The meeting began on a sorrowful note, as Professors Jim Benedix, Bruce Stinebrickner and Dana Dudle spoke about their late colleagues Preston Adams, Darby Morrisroe and Bruce Serlin, respectively.
A motion in support of DePauw's DACA students was presented at the meeting. The motion stated, "DePauw University faculty are united in declaring that we are with Dreamers and DACA recipients. We recognize their enormous role in our communities and families, and their contributions to our schools, workplaces, and shared prosperity as a nation."
DePauw faculty made it clear that they will fight for DACA students and that this issue will not go unnoticed. The authors of the motion want DACA students to know that "… we see you, we value you, and we are ready to defend you."
The motion was submitted by Rebecca Alexander, assistant professor of education studies, Angela Castañeda, chair of the sociology and anthropology department, Jeff Hansen, professor of chemistry and biochemistry, Douglas Harms, professor of computer science, Paul Johnson, assistant professor of modern languages, Glen Keucker, professor of history and Alejandro Puga, professor of modern languages regarded DACA students as "integral members" of the DePauw community and recognized President Mark McCoy's advocacy for DePauw's DACA students. The faculty showed their support of DACA students with a vote to pass the motion.
With the number of hospitalizations rising to 13 over the weekend, Dorian Shager, dean of campus life, and Julia Sutherlin, assistant dean of campus life and director of alcohol initiatives gave a statistics-heavy presentation on DePauw's substance abuse problem and the initiatives they will implement to combat this growing problem. According to the presentation, binge drinking has been steadily increasing at DePauw, unlike colleges across the country, where binge drinking has been declining.
Plans to fix this issue include prohibiting hard alcohol at registered events on campus and allowing approved kegs instead. TIPS training and an awareness campaign have been implemented in order to educate students on the substance abuse problem at DePauw and how they can be active in the process.
DePauw also plans to educate students on the DePauw Safe Community Initiative which can be found in the student handbook and is similar to the Indiana Lifeline law, which was put into law after the death of an Indiana college student whose friends did not call for help when he was dangerously intoxicated.
Further, Karin Wimbley, assistant professor of English, questioned the Humbert bias incident and what sanctions the responsible student received. Alan Hill, vice president for student academic life, told faculty he could not disclose the exact sanctions the student received. Hill did say the student received the "highest level" of sanctions, which includes suspension and expulsion.
The faculty also showed frustration with the lack of transparency from DePauw about the issue when they did not receive an update about how the situation was handled or how the student had been sanctioned.
President Mark McCoy discussed the financial state of the University. "There is no fear of going under and the endowment and property exceeded a billion dollars for the first time," President McCoy said.
President McCoy also gave his support for undocumented students, and the initiatives being put in place for the high risk drinking behavior. Finally, President McCoy touched on the newly found swastika symbol in Asbury Hall.
The next faculty meeting will be Oct. 2.


text:
//-----------------------------------------------------------------------
// <copyright file="TokenCredential.cs" company="Microsoft">
//    Copyright 2013 Microsoft Corporation
//
//    Licensed under the Apache License, Version 2.0 (the "License");
//    you may not use this file except in compliance with the License.
//    You may obtain a copy of the License at
//      http://www.apache.org/licenses/LICENSE-2.0
//
//    Unless required by applicable law or agreed to in writing, software
//    distributed under the License is distributed on an "AS IS" BASIS,
//    WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
//    See the License for the specific language governing permissions and
//    limitations under the License.
// </copyright>
//-----------------------------------------------------------------------

namespace Microsoft.WindowsAzure.Storage.Auth
{
    using System;
    using System.Threading;
    using System.Threading.Tasks;

    #if WINDOWS_RT

    /// <summary>
    /// Represents a token that is used to authorize HTTPS requests.
    /// </summary>
    public sealed class TokenCredential
    {
        private volatile string token;

        /// <summary>
        /// The authorization token. It can be set by the user at any point in a thread-safe way.
        /// </summary>
        public string Token
        {
            get
            {
                return this.token;
            }
            set
            {
                this.token = value;
            }
        }

        /// <summary>
        /// Create an instance of <see cref="TokenCredential"/>.
        /// </summary>
        /// <param name="token">Value of the token credential.</param>
        public TokenCredential(string token)
        {

        }
    }

    #else
    /// <summary>
    /// This is the return type of <see cref="RenewTokenFuncAsync"/>.
    /// A new token and a new frequency is expected.
    /// </summary>
    public struct NewTokenAndFrequency
    {
        /// <summary>
        /// Create a new instance of <see cref="NewTokenAndFrequency"/>.
        /// </summary>
        /// <param name="newToken">The new token credential.</param>
        /// <param name="newFrequency">The new frequency to wait before calling <see cref="RenewTokenFuncAsync"/> again.</param>
        public NewTokenAndFrequency(String newToken, TimeSpan? newFrequency = null) : this()
        {
            Token = newToken;
            Frequency = newFrequency;
        }

        /// <summary>
        /// The new token credential.
        /// </summary>
        public String Token { get; set; }

        /// <summary>
        /// The new frequency to wait before calling <see cref="RenewTokenFuncAsync"/> again.
        /// </summary>
        public TimeSpan? Frequency { get; set; }
    }

    /// <summary>
    /// This type of delegate is used to update the token credential periodically.
    /// </summary>
    /// <param name="state">A state object, which can be of any type.</param>
    /// <param name="cancellationToken">A cancellation token to receive the cancellation signal.</param>
    /// <returns></returns>
    public delegate Task<NewTokenAndFrequency> RenewTokenFuncAsync(Object state, CancellationToken cancellationToken);

    /// <summary>
    /// Represents a token that is used to authorize HTTPS requests.
    /// </summary>
    public sealed class TokenCredential: IDisposable
    {
        private volatile string token;
        private readonly Timer timer = null;
        private readonly RenewTokenFuncAsync renewTokenFuncAsync;
        private readonly CancellationTokenSource cancellationTokenSource;
        private TimeSpan renewFrequency;

        /// <summary>
        /// The authorization token. It can be set by the user at any point in a thread-safe way.
        /// </summary>
        public string Token
        {
            get
            {
                return this.token;
            }
            set
            {
                this.token = value;
            }
        }

        /// <summary>
        /// Create an instance of <see cref="TokenCredential"/>.
        /// </summary>
        /// <param name="initialToken">Initial value of the token credential.</param>
        public TokenCredential(String initialToken) : this(initialToken, null, null, default(TimeSpan)) { }

        /// <summary>
        /// Create an instance of <see cref="TokenCredential"/>.
        /// </summary>
        /// <param name="initialToken">Initial value of the token credential.</param>
        /// <param name="periodicTokenRenewer">If given, this delegate is called periodically to renew the token credential.</param>
        /// <param name="state">A state object is passed to the periodicTokenRenewer every time it is called.</param>
        /// <param name="renewFrequency">If periodicTokenRenewer is given, user should define a frequency to call the periodicTokenRenewer.</param>
        public TokenCredential(String initialToken,
            RenewTokenFuncAsync periodicTokenRenewer,
            Object state,
            TimeSpan renewFrequency)
        {
            this.token = initialToken;

            // if no renewer is given, then the token will not be updated automatically.
            if (periodicTokenRenewer == null) return;

            this.renewTokenFuncAsync = periodicTokenRenewer;
            this.renewFrequency = renewFrequency;

            // when "new Timer(...)" is called, it might call RenewTokenAsync before even being assigned to timer, if renewFrequency is very close to 0.
            // since RenewTokenAsync refers to timer, we need to make sure that before it is invoked, timer is defined.
            this.timer = new Timer(RenewTokenAsync, state, Timeout.InfiniteTimeSpan, Timeout.InfiniteTimeSpan);
            this.timer.Change(this.renewFrequency, Timeout.InfiniteTimeSpan);
            this.cancellationTokenSource = new CancellationTokenSource();
        }

        /// <summary>
        /// Calling Dispose stops the timer and periodicTokenRenewer.
        /// </summary>
        public void Dispose() { this.timer.Dispose(); this.cancellationTokenSource.Cancel(); }

        /// <summary>
        /// This method is triggered by the timer.
        /// It calls the renew function provided by the user, updates the token, and then restarts the timer.
        /// </summary>
        /// <param name="state"></param>
        private async void RenewTokenAsync(Object state)
        {
            try
            {
                var newTokenAndFrequency = await this.renewTokenFuncAsync(state, this.cancellationTokenSource.Token);
                this.token = newTokenAndFrequency.Token;
                this.renewFrequency = newTokenAndFrequency.Frequency ?? this.renewFrequency;
                    // if nothing is given, use previous frequency.
            }
            // only catch the exception when it was caused by this credential object's cancellation token.
            catch (OperationCanceledException ex)
            {
                if (!ex.CancellationToken.Equals(this.cancellationTokenSource.Token))
                {
                    throw ex;
                }
            }
            finally
            {
                // the timer is restarted.
                if (!this.cancellationTokenSource.IsCancellationRequested)
                    this.timer.Change(this.renewFrequency, Timeout.InfiniteTimeSpan);
            }
        }
    }
#endif
}

redpajama_set_name: RedPajamaGithub


Since 2008 Van-Tam has led an international consortium to coordinate the investigation of how the flu virus travels. For the first time ever, 41 volunteers agreed to live together, Big Brother style, in a clinical unit in Cambridgeshire for a fortnight, exposing themselves to the flu virus. The aim was to see if transmission is caused more by large droplets that fly out when you cough and sneeze, or smaller particles, which are referred to as aerosols.
During the four days of the virus exposure period, and subsequent ten days, the flu recipients were constantly monitored by staff, who controlled their contact with each other. They were banned from alcohol, smoking and strenuous exercise to prevent the results being affected.
The investigation is complex, and very expensive. Apart from the intellectual challenge of designing the correct study, the logistical issues are quite immense. Just one quarantine study requires about 18 months of planning and hundreds of staff – and they have three on the go at the moment.
Van-Tam believes that if this research model works, it can be used to study many other related questions about flu transmission in the future. Their findings will help decide the levels of protection for healthcare workers in flu epidemics around the world.
Why not do a language activity based on this cubed story, Flu Study?


text:
These $22 Individually Wrapped Strawberries Are a New Low in Wasteful Packaging
The single berries come nestled inside a Styrofoam sock on top of a pile of faux hay inside of a plastic-covered paper box—a practice that is not only inefficient and wasteful, but gaudy and, frankly, stupid.
by Nick Rose
Feb 8 2017, 6:00pm
Photo via Instagram user emi12.10
Strawberries are good and all, but do they really deserve to be sold for top dollar on an individual basis?
That might sound like the whim of a greedy, despotic leader, but it's actually common practice in Hong Kong supermarkets, where an individually wrapped "Fresh by Air From Japan" strawberry will run you HK$168—a whopping US$21.65.
The single berries come nestled inside a Styrofoam sock on top of a pile of faux hay inside of a plastic-covered paper box—a practice that is not only inefficient and wasteful, but gaudy and, frankly, stupid. A spokesperson from Hong Kong supermarket chain CitySuper argued to Hong Kong Free Press that the packaging connotes the berries' "premium grade, rarity and fragility," which requires "quality protection." The chain also said that the strawberries "are considered as delicacies of limited quantity and they are handpicked to ensure only the highest quality ones are harvested." (That's not to mention that almost all strawberries sold around the world are hand-picked, regardless of their country of origin.)
READ MORE: Pre-Peeled Avocados Are Really Pissing Off Canadian Shoppers
If you're of the opinion that this is a waste of money and resources, you're not alone. More than 8,000 people have signed a petition on Change.org to make that opinion known.
In Hong Kong, you can buy a single strawberry for $168 (about £17.50). O.o #gotosleepjamie pic.twitter.com/5zQlR63XFT
— Dr Jamie Bernthal (@jcbernthal) February 8, 2017
Created by an environmental group called Plastic-Free HK, the petition calls for an end to the wasteful packaging epitomized by the "Fresh by Air From Japan" individual strawberries.
"We are calling on the owner's of every single large grocery store chain in Hong Kong to stop the excessive and unnecessary use of plastic wrap and plastic containers for their produce," the petition says.
Obviously, this isn't just a packaging issue, it's an environmental one. "The plastic pollution problem in Hong Kong and worldwide has reached horrendous levels, with our landfills coming close to capacity, our beaches being overrun with rubbish and our wildlife dying because of mistaking all of this waste for food," the petition goes on to say.
In Pictures: A HK$168 strawberry? HK$4288 apples? Over-packaged 'designer' fruit spark online fury https://t.co/Qzke9LJBRP pic.twitter.com/WW72fPuWy1
— Hong Kong Free Press (@HongKongFP) February 6, 2017
According to the South China Morning Post, just the Styrofoam socks on thousands of fruits in Hong Kong grocery stores weigh in at a total 83 tons every year, and would stretch from Hong Kong to the Maldives if placed in a line.
Plus, paying $20 for a single strawberry—even if it is presented in a nest and flown in from Japan—just makes you a sucker.


Today, the moment to say goodbye to Chile, and to return to the frosty climates up north, has at last arrived. Sitting here at the airport, waiting for my flight to depart, I am in two minds. Sad to leave a place where I have been very warmly welcomed and which I would enjoy exploring more, but at the same time happy to return to the place I've worked hard to make my home.