Investigation of 100G (4x25G) NG-PON2 upgrade using a burst mode laser based on a multi-electrode laser to enable 100 GHz wavelength grid

Investigation of NG-PON2 upgrade to 25 Gb/s line-rate on a 100 GHz grid using a burst-mode transmitter based on a multi-electrode DFB. Compliance with NG-PON2 MSE requirements is shown

10Gb/s low-cost directly modulated multi-electrode laser with suppressed thermal wavelength drift for burst-mode upstream transmission in TWDM-PONs

We report on a novel 10Gb/s low-cost multi-electrode DML employed as a very wavelength stable burst-mode source for upstream TWDM-PONs. 10X wavelength drift reduction is achieved compared to conventional DMLs enabling transmission on 100GHz grid

SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

Background: Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFkB system and TNF signaling. In view of the overwhelming importance of the NFkB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. Principal Findings: BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFkB pathway, but it also diminished the stronger NFkB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. Significance: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies

BV6 induces apoptotic and necrotic cell death in monocytes.

<p>(A) Freshly isolated monocytes were incubated in GM-CSF/IL4 supplemented medium for 1 h with the indicated mixtures of z-VAD-fmk (20 µM), necrostatin-1 (70 µM) and the TNF inhibitor TNFR2-Fc/Enbrel (50 µg/ml). Cells were then challenged overnight with 10 µM BV6 and cell viability was finally evaluated by help of the MTT assay. (B) Freshly isolated monocytes and monocytes cultivated overnight in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by FACS for cell surface expression of membrane TNF and the death receptors TNFR1, CD95 and TRAILR2. (C) Monocytes were challenged with BV6 in the presence of soluble Fc fusion proteins of TRAILR2 (5 µg/ml), CD95 (50 µg/ml) and TNFR2 (Enbrel, 20 µg/ml) or a mixture of them. After 24 h viability was determined using the MTT assay (Left panel). The functionality of the three Fc fusion proteins was controlled in cell death assays with HT1080 and recombinant 50 ng/ml TNF, 4 ng/ml Fc-CD95L and 50 ng/mlTRAIL (right panel). Data shown are representative for three independent experiments.</p

Based on peptide 1 which was synthesized using standard Fmoc solid phase methods on SASRIN™ resin, BV6 (peptide 3) and monovalent (peptide 2) and trivalent (peptide 4) variants derived thereof were synthesized.

<p>Based on peptide 1 which was synthesized using standard Fmoc solid phase methods on SASRIN™ resin, BV6 (peptide 3) and monovalent (peptide 2) and trivalent (peptide 4) variants derived thereof were synthesized.</p

Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death.

<p>(A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in GM-CSF/IL4, iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).</p

GM-CSF and IL4 rapidly induce resistance against BV6-induced cell death in monocytes.

<p>(A and B) Monocytes were cultivated in medium supplemented with GM-CSF/IL4 and were treated immediately or after 1 and 2 days with 10 µM BV6. One day after stimulation BV6 treated cells and a corresponding control sample cultivated without BV6 were analyzed by annexin-V staining (A) and western blotting (B).</p

SMAC mimetic BV6 attenuates TNF- and CD40L-induced proinflammatory signaling and triggers DC maturation.

<p>(A and B) Immature monocyte-derived dendritic cells (iDCs) were generated by cultivation for 7 days with GM-CSF/IL4. To obtain mature dendritic cells (mDCs), iDCs were further treated with Fc-CD40L (200 ng/ml) for 2 days. mDCs were then cultivated in Fc-CD40L–free medium for additional 24 hours with and without 10 µM BV6 and were stimulated for the indicated times with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (A). iDCs were primed overnight with 10 µM BV6 and were then challenged for 5 and 20 min with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (B). iDCs and mDCs were finally analyzed by western blotting to determine the presence of the indicated proteins. The results shown with mDCs are representative of three independent experiments, the results with iDCs for two experiments. (C) iDCs were treated with 10 µM BV6, 300 ng/ml TNF and 1 µg/ml of Flag-CD40L oligomerized with 1 µg of the Flag-specific mAb M2 or as a control remained untreated. After three days cells were analyzed by FACS for the cell surface expression of CD83 and CD86. (upper panel: representative analysis of one individual sample; lower panel: summary of the data of iDCs of 4 independent donors. (D) Cell culture supernatants from “C” were analyzed for the presence of IL12. IL12 production was normalized to the corresponding values of untreated cells. The average of experiments with five independent donors is shown.</p

BV6 and its monovalent and trivalent counterparts moSmM and triSmM induce apoptosis in Kym-1 cells and trigger p100 processing in a variety of cell lines.

<p>(A) Kym-1 cells were seeded in 96-well plates and stimulated the next day in triplicates with the indicated concentrations of moSmM, BV6 and triSmM. After additional 18 h cell viability was determined by MTT staining. (B), Kym-1 cells were incubated in triplicates overnight (16–18 h) with the indicated mixtures of z-VAD-fmk (20 µM), neutralizing TNF-specific mAb Remicade (40 µg/ml), TNF inhibitor Enbrel (50 µg/ml) and the various SMAC mimetics (10 µM) and viability was then again determined by MTT staining. (C) The indicated cell lines were treated with 10 µM BV6 for 6 hours or remained untreated. Total cell lysates were then analyzed with respect to the presence of the indicated proteins by western blotting. (D) Kym-1 and HT1080 cells were primed with 10 µM BV6 for 6 h or remained untreated and were subsequently stimulated for 0, 3, 10 min with TNF (30 ng/ml). Cells were lysed in Laemmmli sample buffer and analyzed by western blotting for the presence of the indicated molecular species. Detection of phospho-IκBα, phospho-JNK and phospho-p38 is indicative for activation of the classical NFκB pathway and the JNK and p38 MAP kinase cascades. An increase in the p52/p100 ratio further argues for enhanced signaling via the alternative NFκB pathway. Detection of tubulin served as loading control.</p

SMAC mimetic BV6 induce no cell death in T-cells.

<p>(A and B) CD4⁺ and CD8⁺ T-cells were isolated by magnetic bead separation and were stimulated for 4–7 days with PHA or remained untreated. Cells were challenged with 10 µM or remained untreated as a control and were finally analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent experiments). p100 processing was determined by western blotting and are shown for one representative experiment (B).</p