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Published in Oncotarget V6I7, Mar 10, 2015
Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors
Oncotarget. 2015; 6:4663-4676. https://doi.org/10.18632/oncotarget.3081
Sebastian Schölch _, Conrad Rauber, Alexandra Tietz, Nuh N. Rahbari, Ulrich Bork, Thomas Schmidt, Christoph Kahlert, Uwe Haberkorn, Mark A. Tomai, Kenneth E. Lipson, Rafael Carretero, Jürgen Weitz, Moritz Koch and Peter E. Huber
Sebastian Schölch1,3,4,*, Conrad Rauber2,3,4,*, Alexandra Tietz3,4, Nuh N. Rahbari1, Ulrich Bork1, Thomas Schmidt2, Christoph Kahlert1, Uwe Haberkorn5, Mark A. Tomai6, Kenneth E. Lipson7, Rafael Carretero8, Jürgen Weitz1, Moritz Koch1,** and Peter E. Huber3,4,**
1 Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany
2 Department of General, Gastrointestinal and Transplant Surgery, University Hospital Heidelberg, Ruprecht-Karls-Universität Heidelberg, 69120 Heidelberg, Germany
3 CCU Molecular and Radiation Oncology, German Cancer Research Center, 69120 Heidelberg, Germany
4 Department of Radiation Oncology, University Hospital Center, 69120 Heidelberg, Germany
5 Division of Nuclear Medicine, Department of Radiology, University Hospital Heidelberg, Ruprecht-Karls-Universität Heidelberg, 69120 Heidelberg, Germany
6 3M, Inc., St. Paul, Minnesota 55121, USA
7 Fibrogen Inc., San Francisco, California 94158, USA
8 Division of Molecular Immunology, German Cancer Research Center, 69120 Heidelberg, Germany
* These authors contributed equally to this work
** These authors share senior authorship
Sebastian Schölch, email:
Keywords: colorectal cancer, pancreatic cancer, TLR7/8 ligand, radiotherapy, immunotherapy
Received: November 29, 2014 Accepted: December 26, 2014 Published: December 31, 2014
In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c+ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.
While considerable progress has been made in the therapy of colorectal cancer (CRC), the prognosis of metastatic CRC is still often unfavorable [1]. The prognosis for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is even worse [2]. Traditional systemic therapies are often unable to eradicate all tumor cells and hence usually cannot provide a cure. In contrast, immunotherapy may be able to definitively eradicate the tumor as it does not target the tumor directly but induces an immune response to tumor antigens in general.
Among the currently most successfully used immunotherapeutics are imidazoquinoline derivatives, most notably imiquimod (Aldara® cream), a selective TLR7 agonist, which can be topically used in malignant and premalignant skin lesions and shows considerable response rates [3]. TLR are pattern recognition receptors involved in the sensing of structurally conserved molecules derived from microbes and therefore predominantly expressed on immune cells, most notably APC (such as DC and macrophages) and effector cells of both the innate and adaptive immune system (such as macrophages, NK cells, B cells and cytotoxic T cells). Upon antigen contact and TLR activation, these cells show increased proliferation, cytokine secretion, costimulatory molecule expression, phagocytic activity and antigen presentation [4]. The agent used in the present work, 3M-011, an agonist for TLR7/8, is soluble in aqueous buffers and can therefore be administered systemically [5,6]. 3M-011 does not activate mouse TLR8, thus acting as a TLR7 agonist only in the murine setting [5].
Tumor vaccines generally aim to induce antigen-specific humoral (B cell-based) or cytotoxic (T cell-based) immune responses. As the response to the antigen is usually insufficient to induce clinically relevant and durable immune responses, numerous adjuvants have been evaluated [7]. TLR agonists strongly increase APC activity and priming of the adaptive immune system, resulting in augmented responses to the vaccine and thus seem to be suitable adjuvants [8]. TLR7 and TLR8 are expressed in plasmacytoid (pDC) and myeloid (mDC) dendritic cells, respectively. Upon activation, mDC produce various proinflammatory cytokines, show increased antigen phagocytosis and processing and prime CD8 and CD4 T cells [9]. pDC produce high amounts of type 1 INF and thus boost the activity of CD8 T cells and NK cells [10].
Radiotherapy is a mainstay in the treatment of many solid tumors including gastrointestinal tumors. Data from multiple cancer models have provided sufficient evidence to propose a paradigm shift, whereby some of the effects of ionizing radiation are recognized as contributing not only to local tumor control but also to local and systemic antitumor immunity [11-14]. The underlying radiation effects include the induction of a pro-inflammatory microenvironment within the tumor, as well as the release of tumor antigens which can subsequently be processed and presented by APC, thus acting as an intrinsic tumor vaccination. As opposed to classical tumor vaccination, which usually induces an immune reaction against a single, tumor-specific antigen, the radiation-induced immune reaction is directed against multiple epitopes. As tumors are immensely heterogeneous, this polyclonal immune response may prevent tumors from evading eradication by downregulating the antigens targeted by the immune system. Both ionizing radiation and pro-inflammatory drugs such as TLR ligands may therefore be able to synergistically contribute to the eradication of tumors by the immune system [15,16].
In the present study, we therefore aimed to investigate the effects of a combination of the TLR7 agonist 3M-011 and fractionated radiation therapy. We hypothesized that the TLR ligand might increase processing and presentation of antigens released by concomitant radiation, therefore acting as an adjuvant to the vaccination effect of radiotherapy (Fig. 1). We demonstrated the effectiveness of the therapy combination and identified the involved immune cell populations and subsequently investigated their role by selective in vivo depletion.
Fig.1: Scheme of putative synergistic effects of the therapy combination.
As previous reports had indicated an involvement of TLR signaling in tumorigenesis [17], we first evaluated the effects of the TLR7/8 agonist on various tumor cell lines. We therefore performed WST proliferation assays on murine and human tumor cell lines of CRC and PDAC and found that 3M-011 had only minimal and divergent effects on tumor cell proliferation in vitro and only at concentrations unattainable in vivo (Supplemental Fig. 1). Of note, there was no direct radiosensitizing effect of 3M-011.
When combined with ionizing radiation, 3M-011 effectively inhibits the growth of syngeneic colorectal and pancreatic tumors and can cause complete remissions
To investigate the effects of the TLR7/8 agonist in combination with classical fractionated photon radiotherapy in vivo, we induced tumor homografts by subcutaneous implantation of CT26 tumor fragments in immunocompetent BALB/c mice. Treatment with 3M-011, fractionated local irradiation (Rx, targeted to the subcutaneous tumor) or the combination of both started when the tumors were established, growing and about 50 mm³ in size. Vigorous tumor growth required the euthanasia of vehicle-treated mice in the control group by day 14 after the start of treatment. Significantly reduced tumor volume was noted in the 3M-011 monotherapy group (p<0.01, Fig. 2A). Unsurprisingly, the 5x 2 Gy radiotherapy regime also reduced tumor volumes (p<0.001) and induced complete remissions in ~20% of the animals. The combination of both 3M-011 and radiation showed complete remission of the tumors in 50% of the mice (p<0.001 vs. control group, p=0.0003 for Rx + 3M-011 vs. Rx alone). The combination group also showed at least additive activity in local control rates, yielding local control in 50% of the mice (х² = 11.2, p<0.01 for two-sided tests). In addition, tumor growth delay (TGD, for 5-fold of the starting volume) was calculated. TGD was 1.58 days for 3M-011 and 6.11 days for Rx (average time to reach 5-fold tumor volume: control 6.89 (4-14) days, 3M-011 8.47 (2-16) days, Rx 13.0 (2-28) days). TGD for the combination treatment could not be calculated as tumors never reached 5-fold of their starting volume, in both monotherapy groups some tumors did not reach 5-fold of their volume during the course of the experiment.
We next tested the treatment combination in a subcutaneous model of pancreatic cancer by implanting syngeneic Panc-02 tumor fragments subcutaneously into C57Bl/6 mice. Treatment was again initiated after the establishment of local tumors. In this tumor model, the effects of the 3M-011 monotherapy were less pronounced than in CT26 tumors. Nevertheless, the combination treatment showed a statistically significant inhibition of growth of the tumors (p<0.05) and the addition of 3M-011 to radiotherapy produced a statistically significant increase in treatment efficacy over irradiation alone (p<0.05, Fig. 2B). TGD (due to the slower growth of Panc-02 tumors as compared to CT26 tumors calculated for 3-fold of the starting volume) was 3.32 days for 3M-011, 1.44 days for Rx and 6.12 days for the combined treatment (average time to 3-fold tumor volume: control 4.35 (2-11) days, 3M-11 7.67 (4-18) days, Rx 5.79 (2-16) days, 3M-011 + Rx 10.47 (2-23) days).
Treatment with 3M-011 and radiation inhibits tumor growth and metastasis in orthotopic pancreatic cancer
In gastrointestinal tumors, the survival of patients is often limited by metastases rather than by the primary tumor itself. Subcutaneous models are mostly models for primary tumor growth. We therefore tested the radio-/immunotherapy combination in a syngeneic, orthotopic mouse model of pancreatic cancer that serves as a model for both local tumor growth and lymph node metastasis [18].
As in the subcutaneous models, we observed a significant reduction in tumor growth achieved by TLR agonist monotherapy (p<0.05). Radiation therapy was delivered to an epigastric target field and also significantly reduced the tumor volume and weight at the end of the experiments (p<0.05). The combination treatment performed significantly better than each monotherapy (p<0.01, Fig. 2C).
The tumors in this mouse model regularly metastasize to mesenteric lymph nodes of the small intestine. The metastatic activity of the primary tumor can be measured by quantification of mesenteric lymph node metastases aligned along the blood vessels. The tumors treated with 3M-011 alone showed a reduced metastatic activity, but failed to reach statistical significance. (33±24% reduction, n=8-10, n.s.). When combined with radiation therapy, this effect was significantly more pronounced (73±21% reduction as compared to radiation alone, 91±7% reduction as compared to the control group, n=8-10, p<0.05 (dual treatment vs Rx alone), p<0.0001 (dual treatment vs. control), Fig. 2D-F).
In addition to the postmortem measurements, in vivo FDG-PET imaging was performed on selected mice from all groups. A decline in metabolic activity in the epigastric target areas was noted in animals treated with both radiation and TLR agonist. Due to the low number of animals subjected to PET imaging, statistical analysis was not feasible (Supplemental Fig. 2).
Fig.2: In vivo therapeutic experiments. A. Growth curves of subcutaneous CT26 CRC homografts. B. Growth curves of subcutaneous Panc-02 PDAC homografts. C-D.Tumor weight and number of mesenteric lymph nodes at day 11 after orthotopic inoculation of Panc-02 PDAC tumor cells . E-F. Exemplary necropsy images (top: abdominal cavity, tumor indicated by white dotted line, bottom: mesentery, metastases indicated by black arrows) of mice with orthotopic PDAC tumors at day 11 after inoculation. Black = Control; green = 3M-011; red = Rx (5x2 Gy photons); blue: 3M-011 + Rx. All values represent mean ± SEM. For clarity reasons, comparisons to the control group are indicated by asterisks without brackets.
3M-011 activates NK cells indirectly via monocyte-derived IL-6
As 3M-011 had minimal direct effects on tumor cells, we concluded that the antitumor effects in vivo were indirect in nature. Previous reports had indicated a pivotal role of NK cells in the treatment with TLR agonists [5], however, the role of other immune cell populations during treatment with TLR7/8 or together with radiation combinations remained unknown.
To further investigate the tumor/immune-cell interaction during treatment with 3M-011, we first performed 51Cr release cytotoxicity assays on human PBMC stimulated with various doses of 3M-011. The TLR ligand dose-dependently increased the cytotoxic activity of human peripheral blood mononuclear cells (PBMC) on HT-29 CRC tumor cells (Fig. 3A). We next aimed to identify the immune cell subpopulations contained within the PBMC fraction responsible for this cytotoxic response. As previous reports indicated a pivotal role of NK cells in the effects of 3M-011 in vivo [5], we performed MACS isolation of CD56+ NK cells from PBMC and evaluated their response to the TLR ligand in cytotoxicity assays. In addition to the expectedly increased baseline cytotoxic activity compared to unselected PBMC, NK cells also showed a marked response to 3M-011. Conversely, the cytotoxic activity and response to 3M-011 of CD56-depleted PBMC was markedly reduced (Fig. 3B).
Previous reports indicated that invariant NK cells could not directly be activated by TLR7/8 ligands although they do express TLR 7 [19]. The authors showed that monocyte-derived DC are required for the activation of NK cells by a TLR7/8 agonist. To test this hypothesis with 3M-011, we first isolated highly purified (>98% purity as opposed to ~90% after MACS purification) CD56+ NK cells by FACS sorting. These cells were subsequently used in the cytotoxicity assays (Fig. 3C) and exhibited increasing target cell lysis with increasing effector:target cell ratios. However, the addition of the TLR ligand to the culture medium did not cause any significant increase in cytotoxicity. As NK cells with relatively low purity (~90%) responded to 3M-011 stimulation whereas highly purified (>98%) NK cells did not, we hypothesized that co-stimulation by a subpopulation in the contaminating cell fraction of the MACS-purified NK cells must be required for NK cells to respond to 3M-011. As previous reports indicated that this contaminating fraction may be cells of the monocytic lineage [19], we artificially contaminated FACS-sorted (i.e., highly purified) NK cells with PBMC-derived monocytes in various ratios and tested the cell mixture for its antitumor cytotoxicity. Fig. 3D shows that a contaminating monocyte fraction of 10% was required to elicit a cytotoxic response to 3M-011.
To test whether this effect was caused by direct cell-cell interaction or an indirect paracrine effect, we incubated monocytes with 3M-011, collected the supernatants und subsequently incubated FACS-sorted NK cells with the monocyte supernatants. Untreated monocyte supernatants were not able to significantly increase the NK cell cytotoxity. However, when pre-treated with 3M-011, the monocyte supernatants dose-dependently stimulated NK cell toxicity (Fig. 3E). ELISA analysis of the monocyte supernatants revealed a marked increase in IL-6 levels upon TLR stimulation, which may explain the increased NK cell activity [20,21].
Fig.3: In vitro assays. A. Dose-dependent induction of human PBMC cytotoxity by 3M-011 in an ADCC assay with HT-29 CRC cells as target cells. B. ADCC assay of human PBMC (left), MACS-enriched CD56+ NK cells (middle) and CD56-depleted PBMC treated with 3M-011 or vehicle. C. ADCC assay of FACS-sorted CD56+ NK cells in various target:effector cell ratios treated with 3M-011 or vehicle. D. ADCC assay of a mixture of FACS-sorted CD56+ NK cells and monocytes in various ratios, treated with 3M-011 or vehicle. E. ADCC assay of FACS-sorted CD56+ NK cells incubated with supernatants of monocytes treated with increasing concentrations of 3M-011. F. ELISA quantification of various cytokines in the monocyte supernatants used in (E). All values represent mean ± SEM.
Tumor-specific cytotoxic T cells are the mediators of the effects of combined radio-immunotherapy
To determine the main effectors of the in vivo antitumor activity, we next depleted wild-type C57Bl/6 mice of either NK cells or CD8 T cells by monoclonal antibody injection (PK136 for NK cells, 53-6.72 for CD8 T cells), as described previously [22,23]. The efficacy and durability of the depletion was repeatedly verified by FACS analyses of mouse blood samples throughout the experiment (Supplemental Fig. 3).
Healthy mice were divided into 4 groups (non-depleted (ND), NK cell depleted (NK), CD8 T cell depleted (CD8) and combined depletion (NK + CD8)) and pre-treated with the depletion mAbs according to protocol (Fig. 4A). After the inoculation of orthotopic pancreatic tumors, each group was then randomly divided into 4 subgroups (vehicle, 3M-011, Rx (5x2 Gy), 3M-011 + Rx) and treated as indicated. Radiation therapy was again delivered in five 2 Gy fractions to an epigastric target field. Tumor inoculation and data collection at the end of the experiment were carried out following the protocol of the orthotopic pancreatic cancer model.
In mice treated with either vehicle or 3M-011 monotherapy, the effects of 3M-011 appeared to be based on the presence of both NK and CD8 T cells (Fig. 4C). In the groups depleted of either cell population, the efficacy of 3M-011 was reduced by about 50-70% compared to non-depleted (ND) mice (p<0.05 for both comparisons). In the double-depleted group, 3M-011 had no measurable antitumor effect.
Interestingly, the anti-tumor effects of the combination of TLR7/8 agonist and radiation appeared to be entirely mediated by CD8 T cells. The efficacy of 3M-011 treatment in the combination group was not affected by NK cell depletion, as the results in the dual depleted group were identical to the results of the CD8 depleted group. In mice depleted of CD8 T cells, however, the addition of 3M-011 to radiation therapy produced no additional effects.
CD11c+ dendritic cell-mediated NK and T-cell priming is required for the effects of the treatment combination
Since both CD8 T cells and NK cells are dependent on previous activation by antigen-presenting cells such as DC, we aimed to investigate the role of DC by selectively depleting these cells in tumor-bearing and treated mice. To this end, we used CD11c.DOG mice, which allow selective and long-term depletion of CD11c+ DC without otherwise affecting the animals [24]. CD11c.DOG are transgenic mice, expressing the diphtheria toxin receptor (DTR) under control of the CD11c promoter. All CD11c+ cells in these mice express DTR and can therefore be selectively ablated by systemic administration of diphtheria toxin (DT). We used the orthotopic Panc-02 model in C57Bl/6 CD11c.DOG mice. To exclude effects of DT on tumor cells, we used wild-type C57Bl/6 mice as controls and treated them with DT in the same manner as the CD11c.DOG mice. Each group of mice (C57Bl/6, CD11c.DOG) was then divided in to the 4 treatment groups (vehicle, 3M-011, Rx, 3M-011 + Rx) and treated as indicated (Fig. 4B). The radiation was again targeted to the epigastrium including the entire pancreas.
Tumor growth and metastasis in DT treated wild-type mice was similar to non-DT-treated mice. Tumor response to both treatment modalities was also consistent with previous results. In CD11c.DOG mice, however, DC ablation led to a near complete abolition of the effects of 3M-011 (Fig. 4D). The effects of radiation alone were also reduced by about 50% (data not shown). Similarly, DC depletion markedly reduced the anti-tumor effects in the combination group of 3M-011 and radiation.
The strong effects of the treatment combination in the previous experiments were indicative of a therapeutically boosted specific immune response. Therefore, we investigated if the treatment combination increased the number of tumor-specific T lymphocytes in vivo. To accomplish this, subcutaneous CT26 tumors were induced in BALB/c mice, treated according to protocol and then T cells were extracted from the spleens to perform ELISPOT assays. The antigens used in this assay were AH1 (a tumor antigen highly expressed in the CT26 cell line) and OVA (control peptide). The ELISPOT assay revealed an increase in CT26-specific T-cells by 3M-011 which was even more augmented by additional radiation (Supplemental Fig. 4).
Fig.4 : A+B: Experimental set-up of the in vivo-depletion experiments. A. NK + CD8 T cell depletion; B. Dendritic cell depletion. Injection algorithms for immune cell depletion are given in the timelines on the right hand sides of the sketches. C+D: Relative tumor weights at necropsy 11 days after orthotopic inoculation of Panc-02 PDAC cells in immune cell-depleted mice. C. Mice treated with 3M-011 (left 4 bars) or 3M-011 + 5x 2 Gy photons (right 4 bars) and depleted of either NK cells (NK), CD8 T cells (CD8), or both (NK+CD8). All bars indicate relative values compared to their 3M-011-naïve controls (left 4 bars show relative values as compared to untreated mice; right 4 bars as compared to the Rx monotherapy group). D. Mice (CD11c.DOG / C57Bl/6) treated with 3M-011 (left 2 bars) or 3M-011 + 5x 2 Gy photons (right 2 bars) and repeated diphtheria toxin injections. Again, all bars are compared to their 3M-011 native controls (left 2 bars: compared to untreated mice; right 2 bars: compared to the Rx monotherapy group). CD8, CD8 T cell depleted; DOG, CD11c-DOG mice; ND, non-depleted; NK, NK cell depleted; Rx, radiation therapy; WT, C57Bl/6 wild-type mice. All values represent mean ± SEM.
3M-011 shifts the intratumoral and systemic cytokine equilibrium to a pro-inflammatory state
To evaluate the effects of the TLR ligand on the intratumoral cytokine profile, tumor tissue of subcutaneous tumors were analysed from mice treated with 3M-011 and ionizing radiation. Changes in the intratumoral cytokine levels of MCP-1, IL-6, INF-γ and TNF-α were measured with the BD Cytometric Bead Array.
The intratumoral levels of all four cytokines were increased by the TLR agonist. For MCP-1, INF-γ and TNF-α, the increase induced by 3M-011 was statistically significant (MCP-1, p<0.0001; INF-γ, p=0.0098; TNF-α, p=0.0024), but the IL-6 increase failed to reach statistical significance (p=0.1451). When combined with ionizing radiation, the intratumoral levels of all cytokines except MCP-1 were further increased (Supplemental Fig. 5A).
Consistent with the increased cytokine levels within the tumors, treatment induced more pronounced systemic cytokine responses. Interestingly, for most cytokines, the systemic increase caused by dual treatment was smaller than the increase caused by 3M-011 monotherapy (Supplemental Fig. 5B). Of note, while INF-γ and TNF-α were not significantly affected by DC depletion, the systemic levels of MCP-1 and IL-6 were significantly reduced after DT administration in CD11c.DOG mice (data not shown).
Here we show that the TLR7/8 agonist 3M-011 is a potent adjuvant to radiotherapy in syngeneic and orthotopic mouse models of colorectal and pancreatic cancer that resulted in marked local and systemic antitumor activity. The rationale of the therapeutic combination was that radiotherapy would provide tumor antigens, while the TLR7/8 agonist would stimulate antigen-presenting cells to process and present these antigens in order to induce and augment an immune response to the tumor (Fig. 1). Mechanistically, 3M-011 activation of APC was essential for NK activation, and both NK and CD8 T cells contributed to antitumor responses to 3M-011 as a single agent. In combination with irradiation, however, CD8 effector T cells dominated the antitumor responses. Nevertheless, DC still had a significant impact on the antitumor response to 3M-011 plus irradiation, since their depletion significantly decreased treatment efficacy. In addition, 3M-011 in combination with irradiation substantially increased intratumoral INF-γ, which in turn may contribute to activation of CD8 effector cells. These mechanisms appear to act in concert to induce a strong anti-tumor immune reaction, which was markedly enhanced by the TLR7/8 agonist over the direct effects of radiation alone.
Tumor cell expression of TLR in carcinomas have been associated with increased growth and invasiveness upon TLR activation [17]. However, 3M-011 did not stimulate the murine or human PDAC and CRC cell lines in vitro, nor did it enhance tumor growth in any of the mouse models. There may be several possible explanations for the differences observed between our study and that of Ochi et al. One reason for the difference might be the different TLR agonists used. Ochi et al. used ssRNA40, E. coli RNA, or Adenine analogs as TLR agonists, which may activate additional signaling pathways than just TLR7/8 as compared to the here used specific TLR7/8 agonist. Another possible explanation may be tumor microenvironment. Although TLR7 was observed in PDAC tumor cells by Ochi et al., they reported that the effects of TLR7 activation on the tumor cells was secondary to TLR7 activation in the surrounding inflammatory cells (28). Subcutaneous tumors (Fig. 2 A,B) are likely to have a different microenvironment than a tumor that develops naturally. Even orthotopic homografts from established cell lines might have a different microenvironment, or might respond differently to their surroundings. Thus, either a different microenvironment or response to the microenvironment might explain the apparent discrepancy between our results and those previously published.
In the subcutaneous tumor models combining 3M-011 with radiotherapy, we observed a significant number of complete remissions despite the relatively low dose of radiation. These effects were confirmed in the orthotopic model of pancreatic cancer, where both primary tumor growth and metastasis were dramatically reduced. In order to characterize the immune cells responsible for these beneficial effects, a series of in vitro and in vivo assays were performed. Using in vitro assays, we were able to show that NK cells were involved in mediating the effects of 3M-011, but required co-stimulation by monocytic cells, which produced significant amounts of IL-6 upon 3M-011 stimulation. This finding is consistent with previous reports indicating low TLR7/8 expression in NK cells and indirect stimulatory effects of TLR agonists on them [19,25].
By in vivo depletion of NK and/or CD8 T cells we showed that 3M-011 monotherapy was dependent on both NK cells and CD8 T cells, both of which contributed approximately 50% of the antitumor effect. Interestingly, when combined with ionizing radiation, the role of CD8 T cells became dominant while the role of NK cells appeared to be negligible. We speculate that this might be the result of an "intrinsic vaccination" effect, which may also explain the beneficial interaction of radiation and TLR stimulation. While the TLR agonist can activate cells of both the adaptive and innate immune systems, its main target cells are DC, which phagocytose, process and present antigens to T cells. Such antigens are released in abundance by radiation-induced cell death, which explains the synergistic effects of the dual treatment. Of note, the tumor sizes in the untreated control groups were not affected by either NK or CD8 T cell depletion, indicating effective immune escape mechanisms of the untreated tumor homografts.
To demonstrate the importance of DC in therapy-induced T cell activation, we used CD11c-DTR transgenic mice. Depletion of DC eliminated the efficacy of 3M-011 monotherapy and significantly reduced the effects of 3M-011 + radiation therapy. The therapy-induced increase of tumor-specific T cells was also demonstrated in ELISPOT assays, which was consistent with data from other groups reporting increased specific immune responses to antitumor vaccination when combined with a TLR7/8 agonist [26].
Cytokine responses within the tumor were more subtle than those observed in plasma, but only within the tumor could induction of cytokines be observed in response to irradiation monotherapy. In contrast, only 3M-011 induced cytokine elevations in plasma. The dramatic cytokine response ("cytokine storm") caused by systemic 3M-011 administration was well tolerated by the animals, but such responses in humans might cause side effects such as flu-like symptoms. Depending on the severity of these side effects, systemic induction of cytokines could result in a dose-limiting toxicity that might require dose reductions or discontinuation of therapy [27]. These considerations suggest that a locally applied TLR7/8 agonist might be preferable.
Imiquimod (Aldara cream) is a topically administered TLR agonist, and the only one that has been clinically approved to date. Imiquimod shows significant activity in many skin conditions including basal cell carcinoma, actinic keratosis (a precancerous form of squamous cell carcinoma [28]) and, off label, melanoma [29]. While topical TLR agonists and radiation combinations have shown promise [30], gastrointestinal tumors are not accessible to topical treatment and require other ways of administration. For the treatment of CRC and PDAC, intratumoral administration (e.g., endoscopically or interventionally) could be a feasible way of administration. Such topical administration would be expected to activate the immune cells in contact with tumor antigens while avoiding excessive systemic cytokine release. In patients with metastatic cancer, an interesting question will be whether local treatment of a single metastatic lesion can induce a systemic immune response. For example, distant metastases in the liver or the skin may be easily accessible to local or even topical treatment. The induction of a systemic immune reaction by treating a single, easily accessible lesion would potentially greatly benefit patients with a yet dismal prognosis and has anecdotally been reported in other tumor entities before [31].
Two recent studies recently published that used a similar approach of combining radiation and another TLR7/8 agonist in a lymphoma [32] and another GI and a fibrosarcoma tumor model [33] are in general agreement with our data, thus independently confirming the combination benefits and supporting the high translational relevance of this therapeutic strategy for a wide range of tumors. Besides demonstrating that this approach may be applicable to additional types of tumors, our data provide conclusive evidence that it is CD11c+ DC that mediate the therapeutic effects via NK and CD8 T cell activation and priming.
In summary, the data presented here clearly show a marked antitumoral activity of 3M-011 combined with ionizing radiation. The effects are mediated by NK and, predominantly, cytotoxic T cells that require activation by DC, which are the main target of the TLR7/8 agonists. These observations suggest that further investigation is warranted.
The human pancreatic adenocarcinoma cell lines Panc-1 and BxPC3, the human colorectal carcinoma cell lines HT29 and HCT-116, and the murine colorectal carcinoma cell line CT26 were purchased from the American Type Culture Collection (ATCC) in 2008. The murine pancreatic carcinoma cell line Panc-02 was generously provided by P. Carmeliet (VIB Vesalius research Center, Leuven, Belgium) in 2010. Panc-1, BxPC3, HT29 and Panc-02 were propagated in RPMI (PAA)) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. HCT-116 and CT26 were propagated in DMEM (PAA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were regularly tested for contamination and authenticated by the DKFZ Genome and Proteome Core Facility via SNP profiling. The last authentication was done in 2012, shortly before the experiments with cell lines were completed.
Proliferation and apoptosis assays
Cell lines were cultured in a final volume of 100 μl/well culture medium in 96 well flat bottom cell culture microplates and incubated overnight. Different starting cell concentrations were assessed. (0/well, 250/well, 500/well, 1000/well). Cells were then treated with 0.1, 1.0 and 10 μM 3M-011 or sodium acetate buffer as a control for 96 hours. Then 100 μl/well WST-1 reagent was added. Cells were incubated in a 37 °C incubator maintained at 5% CO2 for 3 hours, then were shaken thoroughly for 1 min. Absorbance of the samples was measured using a microplate reader at 480 nm. All experiments were done in quadruplicates.
BALB/c and C57Bl/6 mice were obtained from Charles River Laboratories. CD11c.DOG mice were kindly provided by Dr. Günter Hämmerling, DKFZ, Germany. All mice were maintained under specific pathogen-free conditions with a 12hr light/dark cycle and had ad libitum access to standard laboratory diet and water. All animal experiments were evaluated and permitted by both institutional and governmental animal welfare committees (Regierungspräsidium Karlsruhe) and performed in strict accordance to FELASA and GV-SOLAS guidelines.
Subcutaneous tumor models: 1x 105 tumor cells (CT26 or Panc-02) were injected s.c. into syngeneic animals (BALB/c or C57Bl/6). Once the resulting tumors reached a size of 10-12 mm in largest diameter, the tumors were excised and 1x1 mm fragments were implanted subcutaneously into the right flank of BALB/c or C57Bl/6 animals. Tumor volumes were subsequently measured every other day with a caliper, the formula:
, where d is the minor tumor axis and D is the major tumor axis, was used to estimate the tumor volume. Mice were euthanized when tumors reached 20 mm in largest diameter or suffering of the animals became apparent.
Orthotopic PDAC model: 1x 106 Panc02 tumor cells were surgically injected into the head of the pancreas. Mice were euthanized after 11 days or when suffering of the animals became apparent.
Irradiation was delivered in 2 Gray fractions on 5 subsequent days on a Siemens Gammatron unless indicated otherwise. Tumor-surrounding tissue was covered using lead shields.
3M-011 treatment
3M-011 was dissolved in 0,1 M sodium acetate buffer at pH=5.2 and mice were injected i.p. with 200 µl of 3M-011 at dose of 5mg/kg body weight every other day until termination of the experiment. Control group mice received 200 µl of sodium acetate buffer in the same volume and regimen.
For subcutaneous tumors, treatment was initiated when the tumors reached an average diameter of 6 mm. In the orthotopic mouse model treatment was initiated on day 2 after tumor cell inoculation.
The animals were fasted for 4 h prior to PET and kept in an inhalation narcosis with sevorane (0.5 volume-%) and oxygen (flow = 500 ml/min) during the PET examination. The plasma glucose level of the animals was determined using a blood glucose sensor electrode (MediSense) with glucose levels between 100 and 135 mg/dl. A transmission scan was done for 10 minutes prior to tracer administration with two rotating germanium pin sources to obtain cross sections for attenuation correction. The injected activity was 8-9.9 MBq and adjusted according to the weight of the animal which ranged from 14 g to 25 g. PET data were acquired for 20 minutes at 40 to 60 minutes p.i. on an Siemens Inveon scanner (Siemens) using a matrix of 256 x 256 (pixel size 0.3882 x 0.3882 x 0.796 mm). Thereafter, images were reconstructed iteratively using the space alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) applying median root prior correction and were converted to standardized uptake value (SUV) images on the basis of the formula
In vivo depletion of NK cells, cytotoxic T-lymphocytes and dendritic cells
Depletion of NK cells was performed by i.p. injection of 300 μg anti-NK1.1 mAb PK136 (BioXCell) / mouse as described before [22]. Antibody treatment was done 2 d prior to tumor cell inoculation and on days 0,5, and 10 of the experiment (with 0 being the day of tumor cell inoculation). For depletion of cytotoxic T cells, mice were injected i.p. with 200 μg anti-CD8 mAb 53.6-72 (BioXCell) 2 days and 1 day before infection and subsequently on days 0, 4, and 8 as described before [23]. Effectiveness of depletion confirmed in blood on days 0, 4, and 8 by flow cytometry. >98% depletion of CD8 T cells and >95% depletion of NK cells were achieved, respectively. All other groups were treated with the according isotype control antibodies (BioXCell, Fig. 4).
For depletion of DC, CD11c.DOG mice were treated with 8 ng/g body weight of diphtheria toxin (DT) i.p. on day −2, 0, 4, 8, 12, and 16 (with 0 being the day of tumor cell inoculation). CD11c+ cell depletion efficacy was assessed using flow cytometry on days 0, 4, and 8.
Natural killer cell cytotoxicity assay (NKCC)
The bioassay for NKCC was performed using whole blood within 8 hours of collection in a chromium release assay as previously described [34]. The human colorectal carcinoma cell line HT29 was used as target. NK cells were incubated for 16h with indicated concentrations of 3M-011 or cell supernatant prior to NKCC assay. The assay was done in triplicates with 4 hours of incubation time and the relative cytotoxic activity was calculated as follows:
Cell Isolation and Sorting
Peripheral blood mononuclear cells (PBMC) were isolated from freshly obtained blood from healthy donors by Ficoll density gradient centrifugation (GE Healthcare, USA) as described previously [35].
Cell sorting was performed on a FACS Aria II sorting system (BD Biosciences) using MEM-188-APC for CD56, 3G8-Cy5.5 for CD16, and hCD14-FITC for CD14 (all from Biolegend).
Samples were analyzed on a standard BD FACS Calibur flow cytometer (BD Biosciences). Samples were stained according to the manufacturer's protocols for staining of surface antigens in FACS buffer (PBS, 1% fetal calf serum). Samples were analyzed as indicated using the following antibodies: 3G8-Cy5.5 for CD16, MEM-188-APC for CD56, hCD14-FITC for CD14, OKT3-FITC for CD3, PK136-Alexa Fluor 488 for NK1.1, 53-6.7-Alexa Fluor 647 for CD8a (all from Biolegend). Samples were acquired for 3 to 4-colour analysis and analyzed using FlowJo 7.5 software (TreeStar Inc). A total of 500,000–1,200,000 cells were acquired.
Cytokine quantification in mouse serum and tumor lysates
Serum: At day 7 after initiation of the treatment, blood was withdrawn from tumor-bearing mice by puncture of the retrobulbar venous plexus, supplemented with Complete Protease Inhibitor (Roche) and allowed to clot on ice for 30 minutes. The blood was then centrifuged and the serum supernatant obtained.
Tumor tissue lysates: To obtain tumor lysates, tumors were macrodissected and 50 µg of tumor tissue were lysed in 1 ml M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with Complete Protease Inhibitor (Roche) in a TissueLyser II (Qiagen) according to the manufacturers' instructions. After centrifugation, the supernatant was obtained and used for cytokine quantification.
ELISA: The following ELISA kits where used accordingly to manufacturers' protocols: LEGEND MAX Human INF-γ ELISA Kit, LEGEND MAX Human IL-6 ELISA Kit, LEGEND MAX Human IL-12 (p70) ELISA Kit, LEGEND MAXHuman IL-15 ELISA Kit, LEGEND MAXHuman MCP-1/CCL2 ELISA Kit, LEGEND MAX Human TNF-α ELISA Kit (all from R&D) and Human INF-α ELISA Kit, Human IL-18/IL-1F4 ELISA (all from Biolegend).
BD Cytometric Bead Arrays: Proinflammatory murine cytokines (INF-γ, TNF-α, MCP-1, IL-6, IL-10 IL-12p70) were quantified simultaneously using the Mouse Inflammation Cytokine Cytometric Bead Array Kit and CBA software (BD Pharmingen). The kit was used according to manufacturer's instructions on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) on mouse sera and tumor lysates. To obtain tumor lysates, tumors were macrodissected and 50 µg of tumor tissue were lysed in 1 ml M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with Complete Protease Inhibitor (Roche) in a TissueLyser II (Qiagen) according to the manufacturers' instructions.
ELISPOT
To determine systemic tumor-reactive T cell immunity, we isolated T cells from peripheral blood and bone marrow of CT26-bearing BALB/C mice treated with 3M-011 or control. DC were derived from non-tumor-bearing BALB/C mice. We used defined synthetic polypeptides of the tumor-associated tumor antigen AH1 to pulse the DC as described previously [36]. Spot-forming cells were quantified using the KS ELISPOT reader (Zeiss) and CTL ImmunoSpot Analyzer and ImmunoSpot software (Cellular Technology). Spots were measured in the presence of DC pulsed with AH-1 (endogenous tumor antigen on CT-26 tumor cells). Spots measured in the presence of DC pulsed with Ovalbumin where considered as nonspecific background controls. All peptides where obtained from Proimmune, UK. All tests were done in triplicates.
Statistical analysis was achieved by two-tailed student's t-test. All plotted values represent mean ± SEM. p<0.05 was considered statistically significant. Graphpad Prism 6 (Graphpad Software Inc.) was used for statistical analyses and data plotting. Statistically significant values are indicated by asterisks (key: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).
We cordially thank Günter Hämmerling (DKFZ) for the CD11c.DOG mice. We cordially thank Melanie Bernhard, Angela Funk, Andreas Griesbach, Marzena Knyssok-Sypniewski, and Tom Regiert for outstanding technical assistance.
The work was supported by the KFO 227 program (Clinical Research Group 227: colorectal cancer: from primary tumor progression towards metastases (WE 3548/4-1/2) of Deutsche Forschungsgemeinschaft (DFG). This work was supported in part by grants from Deutsche Krebshilfe (106997), DFG National Priority Research Program the Tumor-Vessel Interface (SPP1190), National Aeronautics and Space Administration Specialized Center of Research (NNJ04HJ12G), Kompetenzverbund Strahlenforschung (KVSF, 03NUK004A,C) of Bundesministerien für Bildung, Forschung und Umwelt (BMBF/BMU).
S.S. conceived, designed, coordinated and supervised the project, analyzed the results, and wrote and edited the manuscript. C.R. designed the project, performed the experiments, and edited the manuscript. A.T. performed experiments, analyzed results and edited the manuscript, T.S. contributed the orthotopic PDAC mouse model, edited the manuscript and critically discussed the results. N.R., C.K., D.S., J.K., J.F., U.W. critically discussed the results and edited the manuscript. U.H. conducted and analyzed the PET scans. M.T and K.L. kindly provided 3M-011, critically discussed the results and edited the manuscript. R.C. kindly provided the CD11c.DOG mice and counselled the authors throughout the project. J.W. supervised the project, discussed the results, and edited the manuscript; M.K. and P.H. conceived, designed, and supervised the study, analyzed the data, edited the manuscript, and worked together on the manuscript at all stages.
Potential Conflicts of Interest
M.A.T. is employee of 3M and owns shares of 3M. K.E.L. owns shares of 3M. All other authors declare no potential conflicts of interest.
APC, antigen-presenting cell; CRC, colorectal cancer; DC, dendritic cells; DT(R), diphtheria toxin (receptor); IL, interleukin; INF, interferon; NK cells, natural killer cells; OVA, ovalbumin; PBMC, peripheral blood mononuclear cells; PDAC, pancreatic ductal adenocarcinoma; ND, non-depleted; Rx, radiation; TGD, tumor growth delay; TLR, Toll-like receptor; WT, wild-type.
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21. Luger TA, Schwarz T, Krutmann J, Kirnbauer R, Neuner P, Köck A, Urbanski A, Borth W, Schauer E. Interleukin-6 is produced by epidermal cells and plays an important role in the activation of human T-lymphocytes and natural killer cells. Ann N Y Acad Sci. 1989;557:405–14.
22. Wang M, Ellison CA, Gartner JG, HayGlass KT. Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses. J Immunol Baltim Md 1950. 1998;160:1098–105.
23. Kruisbeek AM. In vivo depletion of CD4- and CD8-specific T cells. Curr Protoc Immunol Ed John E Coligan Al. 2001;Chapter 4:Unit 4.1.
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PII: 3081


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Blog, Info
December 2018 Roundup
Blog, Roundup
December has kept me on my toes. Lots of writing, a few deadlines, some beta reading and even venturing out into the cold.
Nige's Christmas present was a little early – a visit to see Star Wars accompanies by a full symphony orchestra in Leeds. Seeing Star Wars: A New Hope on the big screen, even with the dodgy additional CGI, was exceptional. It reminded me of what I loved about the franchise. Throw in incredible musical accompaniment and it was just superb. A smashing night was had. We also managed to see Aquaman, more on that below.
Korolev Crater
We've been seeing a lot more of Mars than usual, which has been wonderful. Inspiring and incredible. The kind of news we need.
I completed a good solid draft of Codename True Blue. It's a touch shorter than I thought, but I'm really happy with how the idea came together. Miles away from the original, but still ending up well. I'm in a good place to start 2019.
I got through a good number of books in December, from the short story anthology by Joe Hill Strange Weather, to the wonderful Rosewater by Tade Thompson. The Poppy War by R.F. Kuang was a very dark read while The City of Brass was an enjoyable dip into mythology.
I'm a big DC Comics fan. I grew up on them. So while I enjoy the MCU and it's generally good fun, DC films mean a lot more to me because I've read about these people. Batman, Wonder Woman, Superman, Zatanna, John Constantine, even Swamp Thing, they all have a place in my heart.
Arthur Curry? Not so much. There have been the odd attempts at reboots, but I just never got into him. So the fact that this film has changed Aquaman from the Robot Chicken loser we all loved to laugh at to this is incredible figure.
The story hammers through the obligatory origins by mostly making it about his parents a human dad and an Atlantean queen mum before shunting Arthurs backstory to flashbacks. It's then a film about Atlantean society and their deep horror at miscegenation. (Cue the Lovecraft books dotted around in early shots).
There are a huge amount of insulting 'half-breed' versus 'pureblood' comments and as someone who has heard them (or worse) the fact that this is a film about how Arthurs 'mongrel' background makes me exceptionally happy. That background is the only reason he can heal the rifts in the undersea kingdom.
This didn't feel like an accident (which a bit of checking on Twitter proved to be true) and it elevated this campy, fun, silly superhero film a whole other level.
It's a fun film with a very obvious plot you can figure out easily. That said the obligatory reluctant hero is slightly better than the Marvel staple here, we already know he doesn't mind hero-ing (thanks to Justice League), so instead Arthur isn't interested in being king of a place he wasn't raised and doesn't understand. (Someone tell Daenerys this, please.) It's very action-heavy, which is normally a negative strike for me, but it was so nicely made, I enjoyed them. (Excluding the obligatory super CGI end battle most superhero films do now.)
I liked Amber Heard as Mera. She had the thankless task of helping someone who didn't care about her society and she sacrifices everything to help him. It would have been nice for her to be the ruler; smarter, actually socialised as Atlantean, and wants the job, but the actual reason Aquaman gets the job is far less annoying than the first Ant-Man film weak reasoning. I hope she has a royal marriage to actually control the country and Arthur. Dolph Lindgren as Mera's dad was a nice surprise and actually had a wonderful moment or two.
The weakest part for me was Patrick Wilson as his scheming brother Orm who once more was exceptionally bland. I don't know him well, but I remember him from Watchmen where he was also exceptionally bland.
There are some nice extra characters, his dad is played wonderfully by Temuera Morrison and I was happy to see Ludi Lin (hidden under evil makeup), and Michael Beach as Manta's father.
It's also incredibly pretty. From Mera's jellyfish dress to the sweeping ocean depths, it's gorgeous. Beautiful use of space and light and dark.
And no spoilers, but it was great to finally see how Arthur's talking to fish might be a useful skill.
I also saw the new Coen Brothers Netflix film, The Ballard of Buster Scruggs. The film is an anthology of six stories spread across the Wild West. It's a very pretty film with incredibly beautiful cinematography and a lovely score. It's a Coen Brother's film, you can imagine the wonderful stories that slip between farce to pathos.
But what I most noticed, especially after Red Dead Redemption 2, is the lack of women at all or any people of colour, especially Native Americans. While the film does buck a lot of Western movie stereotypes the two biggest culprits still remain. Native Americans are heartless 'savages' and women barely exist.
Forty-five minutes in a sex worker offers her services. That's the first time I noticed a woman speak. Similarly, for Native characters, they only appear onscreen twice and have no meaningful dialogue. They are mentioned a lot though, mostly as bogeymen the other characters fear. They're never allowed to be people.
Ballad of Buster Scruggs
If they catch you, it won't be so good. After they take off every stitch of your clothes and have their way with you, they'll stretch you out with a rawhide, and then they'll drive a stake through the middle of your body into the ground, and then they'll do some other things. And we can't have that.
I'm too deep into an interactive more nuanced take of that era with more black, Mexican, Chinese people etc, so it feels as though this is a film that hasn't progressed past the shortcomings of its roots into anything new.
I was very excited for Patriot Act by Hassan Minhaj and I was right to be. It's a smart show, contemporary politics mixed in with desi commentary and a US-centric, but still valid, worldview. It's obviously not got a long shelf life, but it's so good.
Similarly, Vir Das has a new Netflix special Losing It. It's a little more personal than his 2017 special, but he keeps pushing humour into many topics that aren't talked about with the community such as health and poverty and masculinity.
It's so good to see so much desi content available. We've gone from one or two names to at least ten famous desi actors/comedians now.
I'm still knee-deep in Red Dead Redemption. I still think it's a masterpiece of a game. I wanted to talk more about it but it's too soon. It's a long game and not everyone has finished.
End of the Year 2018
I'm very pleased with 2018. I announced the sale of Goddess of the North to Reuts in January.
My story Samsara came out in Not So Stories in April. I'm incredibly proud of it and how many lovely reviewers related to it and named it their favourite story. The idea that we're colonising a little back ourselves was important to me and it was nice to see Hasan Minhaj saying the same thing.
America is becoming South Asian.
Tan, America is becoming South Asian. Yoga, meditation, chai tea at Starbucks, us.
I judged an award. It was really fun to read all of the works and discuss their merits. It's something I'd like to do again.
I started officially mentoring someone for SFWA! They began a programme and I realised I might be able to help. I have a lovely mentee who is incredibly talented and will be wowing the world with more of their writing into 2019.
I attended Eastercon and met up with some of my Clarion West buddies as well as meeting new authors. It's a good con & I'm hoping to attend again in 2019.
As for representation, 2018 has been a good year. Sacred Games was Netflix's first Indian original series and it was great. I need more of these, please. Mowgli. Well, it wasn't as awful as the Disney live action and had more than one desi actor. The Indian Detective was a delight. Doctor Who being set in Sheffield and having Yasmin Khan. Riz in Venom, Rami as Freddie, Karan Soni in Deadpool 2, everyone in The Magicians, plus so much more. Let's hope next year continues this trend.
There has been great media in general, from the fifty-three books I read this year, to programmes such as The Expanse and Killing Eve, Dirty Computer from Janelle Monae with Tessa Thompson (who also wowed me in Annihilation, Sorry to Bother You and Westworld.)
I'll talk more about goals in January, (when I figure some out). On to 2019!
November 2018 Roundup
2015 Roundup
Top 10 Fantasy Writing Tips From 'Game Of Thrones' Author George R.R. Martin
The End Has Come: An Interview with Will McIntosh

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Home/Quake Champions Review
Quake Champions Review
Posts on : October 4, 2018 / Posts by : Gunner Davidson	/ Reviews
Quake Champions is the latest game in the Quake series, and it was released in 2017. It continued with some aspects of the series and also changed some parts of the game. These changes received a mixed reception with some Quake fans liking them and others not so much. Change often does receive a mixture of support and dislike, more details of the changes will follow.
It's Quake but not as we know it
Quake Champions was the first official game in the series since Quake 4 way back in 2004. In the meantime there have been unofficial MODS and other games that are really similar. The developers id Software had
previously stated that Quake Champions would not be a MOBA game and this concerned some fans that the game would be won by the players with the biggest weapons and the thickest armor instead of the the ones with the most most skills.
The bigger they are, the harder they fall
Quake Champions does give players different weapons and amounts of armor. In theory the players that have the biggest weapons and the strongest armor have the best chance of winning. However the changes are not as one sided as you belief they are at first. For the players with the most armor and the most weapons also have the largest hit boxes. In other words a good player with an accurate aim can score more hits on a more powerful foe.
There are some surprises
The thing with Quake Champions is that some of the characters have new or secret weapons, which means that you can think that you are doing well against Sorlag when a surprise acid attack kills you. Once you have been killed by weapons or tactics you were not expecting the game becomes more of an even contest, as it is your skill and speed pitted against those of your opponent. The problem is that you may get killed several times in two or three hours while you get used to the game.
Take time to get used to the changes
Typically if you have been used to playing any of the earlier Quake games over a number of years you are less likely to like Quake Champions than if you never played any of the others. For old hands at Quake it was the purity of it's MOBA that placed it among the best of the first person shooter games. Champions has changed the game yet it has not completely ruined it.
Old hands at this game should have patience with this game, and once they get used to it then they should be able to show how skilled they are. Just allow time to learn the new controls.
New players, especially those with experience of playing other games like Overwatch will probably like the game more from the first time that they play it.
The graphics, animation and gameplay are of the high standards you would expect from the Quake series as is the multiplayer mode.
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Stroyent is a gaming blog for any serious Quake players and fan of other FPS games. Bringing you all the latest updates, reviews, guides, walkthroughs and more.
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text:
from helpers import *
from basecommands import simplecommand

ac_permission  = "utils.ac"

ac_defaultkey  = ","
ac_keys        = open_json_file("adminchat_keys", {})

ac_toggle_list = []
ac_prefix      = "&8[&cAC&8]"

def adminchat(sender, msg):
    name = "&7{unknown}"
    try:
        name = sender.getDisplayName()
    except AttributeError:
        name = sender.getName()
    broadcast(ac_permission, "%s &9%s&8: &b%s" % (ac_prefix, name, msg))
    # Needs something here like fine(message) to show up in the logs when you use ackey, but fine doesnt work for some reason. It did on the server with /pyeval (not show up on console, but show up in logs nevertheless)

# ac toggle
@hook.command("act")
def on_act_command(sender, args):
    if sender.hasPermission(ac_permission):
        p = sender.getName()
        if p in ac_toggle_list:
            ac_toggle_list.remove(p)
            msg(sender, "%s &aAC toggle: off" % ac_prefix)
        else:
            ac_toggle_list.append(p)
            msg(sender, "%s &aAC toggle: on" % ac_prefix)
    else:
        noperm(sender)
    return True

@hook.command("ac")
def on_ac_command(sender, args):
    if sender.hasPermission(ac_permission):
        if not checkargs(sender, args, 1, -1):
            return True
        adminchat(sender, " ".join(args))
    else:
        noperm(sender)
    return True

def get_key(uuid):
    key = ac_keys.get(uuid)
    return key if key != None else ac_defaultkey

@simplecommand("adminchatkey",
        aliases = ["ackey"],
        senderLimit = 0,
        helpNoargs = True,
        helpSubcmd = True,
        description = "Sets a key character for adminchat",
        usage = "<key>")
def adminchatkey_command(sender, command, label, args):
    key = " ".join(args)
    uuid = uid(sender)
    if key.lower() == "default" or key == ac_defaultkey:
        del ac_keys[uuid]
        save_keys()
        return "&aYour adminchat key was set to the default character: '&c%s&a'" % ac_defaultkey
    ac_keys[uid(sender)] = key
    save_keys()
    return "&aYour adminchat key was set to: '&c%s&a'" % key

def save_keys():
    save_json_file("adminchat_keys", ac_keys)

@hook.event("player.AsyncPlayerChatEvent", "low")
def on_chat(event):
    sender = event.getPlayer()
    msg = event.getMessage()
    if sender.hasPermission(ac_permission) and not event.isCancelled():
        key = get_key(uid(sender))
        if sender.getName() in ac_toggle_list:
            adminchat(sender, msg)
            event.setCancelled(True)
        elif msg[:len(key)] == key:
            adminchat(sender, msg[len(key):])
            event.setCancelled(True)

redpajama_set_name: RedPajamaGithub


The installation of the scaffolding and shoring of Unity Temple's south roof slab is complete. Even though the staff was accidentally trapped downstairs by a rogue crossbar, it was a very interesting process to watch. It is also a significant milestone. Now that the roof is supported, demolition of select segments can begin.
The entrance hall is now a thicket of shoring posts. They have been "shrouded" in white muslin so people won't bump into the posts and hurt themselves, but here's a look at the bare bones. If you look carefully, you can see how Safway used steel I-beams to bridge the loads over the old heating tunnels.
While C.P. and the Safway engineer reviewed the scaffolding and shoring, Zera continued to remove plaster from the underside of the roof slab. They also submitted two concrete mix designs to C.P. for approval.
Next: a trip to the glass restorer's studio.
Oh, good thing no one was harmed in that accident. The crew should be more careful next time! Anyway, why did you have to demolish the selected segments of the temple? Its roof must be very durable since you didn't have to remove it! How many days did you make the steel support?
The important to installation of the scaffolding and shoring are durable and safe.


We are glad to announce that we have completed testing our latest inline releases of Norton Identity Safe v3.5.1.677 for Android with added new features and bug fixes.
The version number is 3.5.1.677 for Android.
Are there any updates to the list of apps that can be accessed with the Android ID Safe app?


text:
mc-community
DAN SIMMONS: The hunt for new possibilities
It's a new year with new opportunities and possibilities. Now it is time to set our priorities and make plans. My friend Bill Wilson did just that this past year as he set his sights on an African Cape Buffalo safari in Zimbabwe.
Did you miss Santa Claus in Beatty? Here's how you can see him again
By Richard Stephens Special to the Pahrump Valley Times
Santa and Mrs. Claus will be passing out gifts at the Beatty Volunteer Fire Department's Santa Night at the Beatty Community Center on Friday, Dec. 16, starting at 6 p.m., where there will also be hot cocoa and coffee and baked good treats for all.
NyE Communities Coalition: A 20-year retrospective
December 9, 2022 - 7:00 am December 9, 2022 - 7:00 am
The CEO and one of the founders of the nonprofit reflects on two decades of serving the community ahead of the agency's 20th anniversary.
This Pahrump 10-year-old will give dozens of bikes to kids this Christmas
December 8, 2022 - 12:26 pm December 8, 2022 - 12:26 pm
Avery Sampson's 3rd Annual Bikes for Christmas drive is only a few short of its goal of 60 bikes this year.
PHOTOS: Bowl-A-Thon raises nearly $5k for Wounded Warrior Project
December 8, 2022 - 9:15 am December 8, 2022 - 10:55 am
The Nugget Bowl inside the Pahrump Nugget Hotel Casino was packed with patrons on Sunday, Dec. 4 taking part in more than just an afternoon of fun. Here's what you missed.
Pahrump Senior Center hosts Thanksgiving dinner
By Selwyn Harris Pahrump Valley Times
The Pahrump Senior Center served more than 200 meals on Wednesday for its annual Thanksgiving Dinner this year.
PHOTOS: See photos from the Community Thanksgiving Dinner
Linda Wright, committee chair of the Pahrump Holiday Task Forces, aid it was definitely a Thanksgiving to remember.
ICYMI: See photos from the Community Christmas Tree lighting
December 1, 2022 - 11:50 am December 1, 2022 - 11:50 am
It's the most wonderful time of the year, with the holidays officially in full-swing and the community ready to embrace all of the jolliness that the season brings.
Nascar tickets up for grabs at Bowl-A-Thon
This Sunday, the Pahrump Nugget Bowling Center will be taken over by supporters of America's heroes during the Wounded Warriors Bowl-A-Thon and event organizer Denise Flanagan is excited to announce the addition of an extraordinarily generous donation to the cause.
Bowl-A-Thon to benefit Wounded Warriors
November 25, 2022 - 7:00 am November 25, 2022 - 7:00 am
Hosted by local veteran Denise Flanagan, all of the money raised at the Bowl-A-Thon will be donated to Wounded Warrior Project, an organization that supports veterans.
PHOTOS: Quilts of Valor
November 23, 2022 - 12:49 pm November 23, 2022 - 12:49 pm
Comfort and healing bestowed upon local veterans.
Beatty to add fire hydrants
BEATTY — Some areas here will be safer thanks to the decision of the Beatty Town Advisory Board to fund new fire hydrants in three locations in town. The new hydrants will be located in the general areas of North C Avenue and West North Avenue, North E Avenue and West Lisle Avenue, and South B Avenue and West Crowell Avenue.
PHOTOS: Retired military honored on Veterans Day
Here in the Pahrump Valley, where thousands of former military service members call home, Veterans Day is always marked with proper ceremony thanks to the efforts of local veterans' groups.
Hospice launches program to feed homebound veterans
The Food Assistance Program will launch on Veterans Day, Friday, Nov. 11, and will be available to veterans living in Pahrump who are homebound, regardless of it they are a Nathan Adelson Hospice patient.
Community Thanksgiving Dinner seeks volunteers —here's how to help
The Community Thanksgiving Dinner is scheduled for Thursday, Nov. 24 from 11 a.m. to 2 p.m. at the NyE Communities Coalition, 1020 E. Wilson Road


text:
Miejscowości:
 Fago – gmina w Hiszpanii

Osoby o tym nazwisku:
 Nicola Fago – włoski kompozytor

redpajama_set_name: RedPajamaWikipedia


If you would like to be removed from this mailing list, ask your son or daughter to return to Wolverine Access and navigate back to the same page where he/she registered you initially (Student Business > Student Center > Parent/Family Authorization). Once there, your student can review your e-mail account authorization settings. Your inclusion on mailing lists can be edited or your account can be removed altogether by your student. Unchecking the "Alerts, Bulletins" checkbox will immediately remove you from all future Family Matters mailings. However, please be aware that being removed from this group would also mean that you would not receive any future targeted announcements about the University's response to unexpected or emergency situations.


I'm thrilled that my new collection of poetry, We Come Elemental, has been nominated for a Lambda Literary Award in poetry. It's a huge honor to be a finalist among poets I count as important influences, mentors, and friends. Yay!


Wonkhe collaborates with a range of supporters which share our commitment to working with UK higher education. We work together on projects, events and sharing insights about HE.
Wonkhe's partners from across higher education include agencies and associations as well as individual institutions.
Wonkhe works with a select group of professional association partners and we help them support their members.
For more information about our partner and supporter schemes, contact the team.


Washington, D.C. July 17, 2013: "The verdict in the Zimmerman case seems to be enraging both liberals and blacks. Now they want to have him tried again by the Obama people but this will not happen. Here are two comments from readers of the Guardian's articles. Soon enough, there will be rioting in the inner cities and the resulting looting of shops. No one seems to care if the CIA or the Army blows up a Pakistani day care center on the mistaken impression it is a training center for terrorists but I suppose it depends on whose ox is gored.
"No wonder Snowden doesn't think he's going to get a fair trial.


Unibg Security Lab is the Computer Security Team at Università degli Studi di Bergamo.
Its work focuses on several areas in computer science such as systems security (UNIX/Linux security), mobile security (especially Android security and malware analysis), information systems, database technology (data warehouses, workflow management systems), Web, emerging technologies and information security (security for databases, access control, secure reputation in P2P networks, data outsourcing and privacy).
The team is often involved in european projects, and is currently working on cloud security technologies with the EscudoCloud project. The recent work on Android security allowed the team to obtain two Google Awards during the last three years. Last but not least, some of the members usually take part in CTF competitions.
We are always looking for smart, hardworking thesis students. If you are interested in computer security, come talk to us!
The source code of our open source projects is available at: https://github.com/unibg-seclab/ .
Star the projects on GitHub to receive updates on future releases.
The APM project won a Google Award in Winter 2016 batch.
The SeSQLite project won a Google Award in the Winter 2014 batch.


text:
Erdogan Blasts US at White House. Was That Good for NATO?
By Kevin Baron
"I'm a big fan of the president," Donald Trump said of his guest Recep Tayyip Erdogan, the autocratic Turkish leader whose military and allies recently chased American troops and partners from northern Syria. Then Erdogan stepped to the microphones at Wednesday's White House press conference and laid into the United States.
Standing in the East Room, the Turkish leader dismissed the U.S. approach in Syria, branded U.S.-backed Syrian Kurdish fighters as "terrorists," rapped Congress over proposed sanctions and Armenian genocide measures, and said the Pentagon's refusal to supply Patriot missile batteries was an "injustice" that forced Turkey into Russia's arms.
All these issues can be resolved through dialogue, Erdogan said, painting Turkey's relations with the U.S. and NATO as better than they actually are, and showing no signs of budging on any of them.
This was exactly the moment senior Republican and Democratic national security leaders tried to avoid. But it was a moment many U.S. and NATO leaders think was necessary.
For weeks, members of Congress from both parties had called on Trump to uninvite the Turkish president, to deny reward to a leader who increasingly has become a pariah of NATO and the democratic community. Instead, Trump invited Sen. Lindsey Graham, R-S.C., and others to meet Erdogan in the White House. Senate Foreign Relations Committee Chairman Bob Menendez, D-N.Y., turned down the invitation and instead delivered a floor speech noting 10 grievances with Turkey and condemning Trump for staging Erdogan's visit.
Menendez and other critics are furious at Trump for pulling U.S. troops from northern Syria last month at Erdogan's insistence, which they argue ceded a strategic foothold to Russia and Syria, allowed Turkey and Turkish-backed forces to seize Syrian land, and opened U.S.-backed Kurdish fighters to attack. They are angry that Turkey has cozied up toward Moscow, bought the Russian S-400 anti-missile battery, and willfully disqualified themselves from the network of countries that fly the advanced F-35 fighter jet. And, they argue, Erdogan and his regime have strayed into autocracy, with a long list of human rights and political power abuses that could justify ejecting Turkey from NATO and other in international institutions.
Yet other security leaders say the visit may help keep a bad situation from getting worse.
NATO members, U.S. military leaders, and some lawmakers and appear to be doing all they can to keep Turkey — and the Turkish military — close to Washington, Europe, and the alliance, hoping to prevent Erdogan from moving further down its path toward Russia.
Fighting ISIS, or not
Trump, long known for praising authoritarian leaders, was no less effusive for Erdogan and his government. "Turkey, as everyone knows, is a great NATO ally, and a strategic partner of the United States around the world," he said. "We're grateful to President Erdogan and to the citizens of Turkey for their cooperation in the constant struggle against terrorism. He fights it like we do."
Yet Turkey and Turkish forces do not fight like U.S. troops, it seems. In the weeks since the incursion began, U.S. intelligence, independent human rights watchers, and journalists have documented potential war crimes, including direct attacks on civilians. Trump's only nod to these allegations came in his prepared opening remarks: "We've assured each other that Turkey will continue to uphold what it's supposed to uphold."
Among those trying to keep Turkey closer to NATO than Moscow is alliance Secretary General Jens Stoltenberg, who is in Washington this week to meet Trump and leaders of the global counter-ISIS coalition. He has had the unenviable job of walking the line between Ankara and the rest of the alliance, even as some wonder whether NATO should cut Turkey loose.
"There are different views, but we agree on several things, including that Turkey has legitimate security concerns," Stoltenberg said in October. Last week in Berlin, he added, "I have expressed my deep concern about the consequences of the incursion into northern Syria, and I also stated clearly that we must not jeopardize the gains, the progress we have made in the fight against ISIS."
The world will be watching how NATO leaders treat Erdogan in next month's London summit, looking for cracks in the alliance's armor.
Lawmakers disagree
The press was allowed to watch the opening minutes of Erdogan's Wednesday meeting with Trump and members of Congress. Some lawmakers kept their remarks cordial.
"As previously stated, we do want to remain very strong allies in NATO. You are very important to us. So we want to bring you back into that fold. We want to discuss the S-400," said Sen. Joni Ernst, D-Iowa.
Added Sen. Tim Scott, R-S.C., "My goal here and I think all of our goal here is, at the end of this meeting, we're in a better position where we're better allies; where we understand exactly what's going to happen with the S-400; so it's not — so Turkey is heading in the direction of the United States, not heading the direction of Russia."
To that, Trump said, "Right. Very good. It's true."
But then Sen. Ted Cruz, R-Texas, piped up. "The Kurds have risked a lot to stand with America to fight against our shared enemies, and there is very real concern that we do not want to see Turkey engaged in offensive action against the Kurds." He called that and the S-400 buy issues that are "real and significant."
Erodgan quickly asked to interject, and Trump permitted him. "First and foremost, we have to make a distinction here — the Kurds and the terrorists." He detailed Turkey's own Kurdish population and his work to take in refugees, saying, "I assume the ones that you're referring to as Kurds are either PYD or YPG. These are terrorist organizations and they are the offshoots of the PKK. And I would like to submit to your party some documents, specifically."
Erdogan continued to cite Turkey's refugee assistance efforts for Kurds. Trump did not counter Erodgan's SDF characterization. A reporter shouted a final question about the S-400, and Trump thanked the press and ended the photo op.
"I project that we will work something out with Turkey. I think it will work out fine. Okay? Thank you. Thank you. Thank you."
Related podcast:
By Kevin Baron // Kevin Baron is the founding executive editor of Defense One. Baron has lived in Washington for 20 years, covering international affairs, the military, the Pentagon, Congress, and politics for Foreign Policy, National Journal, Stars and Stripes, and the Boston Globe, where he ran investigative projects for five years at the Washington bureau. He is a frequent on-air contributor and previously was national security/military analyst at NBC News & MSNBC. Baron cut his muckraking teeth at the Center for Public Integrity and he is twice a Polk Award winner and former vice president of the Pentagon Press Association. He earned his M.A. in media and public affairs from George Washington University, his B.A. in international studies from the University of Richmond, and studied in Paris. Raised in Florida, Baron now lives in Northern Virginia.
https://www.defenseone.com/ideas/2019/11/erdogan-blasts-us-white-house-was-good-nato/161302/


Eala, eal, ai, swan. 'Eala bhan,' fair swan; 'eala-gheal,' white swan; 'eala ghlas,' grey swan; 'eala-dhonn,' brown swan, cygnet.
There is no bird of passage so welcome in the Western Isles as the swan. Its size, its beauty, its mysterious, plaintive melody, give it a semi-sacred character in the eyes of the people. It is interesting to see swans feeding, and varieties of small ducks, chiefly teal, jerking in and out among them, busily picking up the animalcula and fragments brought up by the swans. The swans take no notice of the ducks, but treat them with dignified indifference, even when the ducks pass under their bills and necks.
In severe winters swans come in large flocks to the Western Isles. When the freezing of the water seems imminent the swans will flap the water with their wings to keep an open space, taking the work in turn. When they are frozen out of the lake they betake themselves to the estuaries of the sea. Swans, like geese, fly in wedge-shaped flocks, often at a high altitude. But even when the flock is only an indistinct haze their striking melody fills the air. To see several hundreds of these beautiful birds together, as they sail rather than fly overhead, is a sight one would not willingly forget, while their liquid voice is like the music of the long-ago echoing through the cloistered cells of memory.
Thug mi a dh' Eirinn.
That took me to Eire.
Gu bhi gi, gu bhi go!
Gu vi gi, gu vi go!
Voice of the swan on the lake.
Although the singing of the swan is not generally acknowledged by ornithologists, it is a widespread and an old belief. Several of the Latin poets speak of it, and mention of it is also to be found in German and Russian authors. Cf. Müllenhoff's Altertumskunde, where an interesting account is given of the song of the swan.
When all the others have forsaken her.
[paragraph continues] This is true to nature.
When she sings a melody.
To see if my fame will cleave to me.
[paragraph continues] The swan was vowed upon in England also:--'Edward vowed on the swan.'--Green's History.
ridge of the gallows, a few hundred yards to the south. In Iona, at 'Port nam marbh,' port of the dead, where the dead were landed for burial in the holy isle, there is a raised platform called 'eala.' The platform is in the form of an altar, and the dead were carried three times sunwise round it and placed upon it before burial. All the stones known to me called 'eala' were places of sanctuary. The poetess Mairearad NicLachuinn has 'dhol air tir air an Ealaidh,' to land at the 'eala,' in Iona.
Ear, east. The old people paid much attention to the orientation of their dwellings and temples.
With its head in the east.
Eararadh, seeking, searching. 'Air eiriridh,' on the search; 'eiriridh maidne,' morning search; 'eiriridh chloimh,' wool-seeking; 'eiriridh dhaoine,' seeking people.
'S ge d' bhiodh i pronnadh eiririch.
'S e Domhull a rinn a bhanais!
As if tramping parched corn.
'Twas Donald made the wedding!
That made the famous wedding!
[paragraph continues] In Shetland the parched grain and the meal from it are called 'burstin,' probably from the tendency of the grain to burst in the process of drying.
Earasaid, a wide mantle that used to be worn by women in the Highlands. Occasionally it was made of tartan, but generally of 'iomairt.' The 'earasaid' is mentioned in a song sung by Boswell at Rararsay. The subject of the song is Prince Charlie, over whom Highlanders lost their heads and their hearts.
I must arise and wield the claymore.
But is in Killpheadair a band of them.
Earc, a heifer, cow, beast of the cow kind; 'earc iuc,' notched cow, from 'earc,' a cow, and 'iuc,' a notch, possibly applied to the Caledonian cattle.
Earnach, arnach, red-water in cattle, red pleura, bloody flux.
Earrlait, rich soil, ground manured one year and productive the next, productive animals, prosperous undertaking.
Eidhion, iadhain, eidh-shlat, iadh-shlat, and eidhion mu chrann, ivy. 'Iadh-shlat' is more often and more correctly applied to the honeysuckle.
Ivy is one of the many sacred shrubs of the Celts. It is used as a protective for milk, milk products, flocks, and by lovers as an emblem of fidelity. An old man in Uist said that he used to swim to an islet in a lake in his neighbourhood for ivy, woodbine, and mountain ash. These, sometimes separately and sometimes combined, he twined into a three-plied 'cuach,' ring, which he placed over the lintel of his cow-house and under the vessels in his milk-house, to safeguard his cows and his milk from witchcraft, evil eye, and murrain.
The term 'iadh-shlat' is used by old people, and occurs in old poetry.
'S fath a broin an cos nan coilltean.
And the cause of her sighs in the depths of the woods.
A Dhuarain c'uim a sheas!
A Ghoill c'uim a thuit!
Mar iadh-shlat mu stoc aosda.
Duaran, why didst thou stand!
Goll, why didst thou fall!
As the twining-wand around the aged tree.
This fragment was taken down in 1860 from Kenneth Morrison, Trithion, Minginis, Skye. Kenneth Morrison was then blind and old, but he remembered many beautiful and rare old poems with more or less completeness. These he heard when a boy at the 'ceilidh,' of which he gave many graphic descriptions.
Eigir, Aegir, a god, a deity, a king. In Norse mythology Aigir is king of the sea, god of the ocean. In Celtic mythology he is king of the dwarfs, god of the misers.
In Arran, 'iasg eigir' is a small fish, a dwarf fish, and 'iasgach eigir' is a poor fishing, whether for the night or for the season. In Barra, 'ubh eigir' is a small egg, a dwarf egg, while 'uibhean eigir,' dwarf eggs, is a term applied to the eggs of the smallest sea and land birds.
'Eigir,' wrongly 'seigir,' is applied to the little gull, an occasional visitor, and more commonly to the kittiwake, the smallest permanent British gull.
Besides 'Lioc a Eigir' in South Uist, there is 'Laimrig Eigir,' landing-place of Aigir in North Uist, and there are in Benbecula 'Loch Eigir,' lake of Aigir, 'Eilean Eigir,' island of Aigir, 'Sgeir Eigir,' the reef of Aigir, and 'Iol Eigir,' the fishing-place of Aigir.
The lake is small and full of small brown trout. The island is merely a rock on which some grass grows. It is the resort of the black-headed gull and of the Arctic tern in their season.
An old 'clachan,' path of stepping stones, connects the rock with the shore, and indicates that the fishing-place was known to people in olden times.
Am fad 's a bhithinn beo.
As long as I would live.
and other cities--in order to add their land to the already extensive lands of tacksmen, one of these being the parish minister.
Torarnis, Torrarnis, Torrannis, is the point of Thor, the point of the thunderer. There are two places of this name in South Uist, and in the neighbourhood of one another, both famed for bere.
There are no nuts there now, nor anywhere in Uist, nor bushes nor trees of any kind nothing but long reaches of sessile sand here and there overlying long stretches of compacted peat. Hazel-nuts in great quantities have been found in Uist lying on the glacial rock. In many places round the west side of the Outer Hebrides the remains of trees of various sizes have been found at low-water embedded in the hard peat moss underlying pure sand.
With the great sea round its middle.
[paragraph continues] This prophecy is to some extent verified, for vast tracts of lands and woods, and in some places the remains of dykes, houses, and churches, can be seen along the coast at low-water.
Carlyle speaks in Heroes and Hero-Worship of the boatmen on the Yorkshire Ouse calling out when the river is in flood--'Eager is coming! Eager is coming!' 'Eager' is also known on the Severn. In this case the idea is that of the Norse giant. A deity of this name is also god of the muses in Celtic mythology.
Eoir, spell, charm, incantation. 'Eoir' in Lewis, 'eolas' in Uist.
Eolas, eoilse, eoisle, a spell, charm, incantation, magic, exorcism, knowledge.
Eorlain, earlain, arlain, floor, bottom, lower part, a glen that slopes to a narrow compass, from 'earr,' limit. The three planks on each side of the keel of a boat are called 'eorlain,'--'eorlain na h-eithir'--bottom of the boat, in this case from 'earrlain,' keel.


text:
EVOLUTION OF THE AUTOMOBILE.
Square, London. Fig. 6 shows a carriage invented by Burstall and Hiel, which attracted a great deal of attention. It was probably the most complete and perfect mechanically of any invention that had been made up to that time.
Fig. 6. Burstall and Hiel's Steam Carriage, Made Prior to 1825.
Fig. 7 shows a carriage invented by Squire and Maceroni, who had been for a long time in the service of Goldsworth Gurney, one of the most noted experimenters of his day in steam propulsion. A number of carriages were made by these workers, on designs similar to Fig. 7, and it is said that they ran at a high rate of speed, probably ten miles per hour.
Fig. 8 illustrates an invention that is interesting from the fact that
Fig. 7. Steam Carriage, Made by Squire and Maceroni.
it was to be operated by compressed air, and perhaps was the first effort to utilize this form of stored energy for the propulsion of vehicles. It was not a success, but its failure was due to the fact that the inventor labored under the delusion that the laws of nature could be circum-


When the manager of a North Carolina Walmart heard about Shelli Tench's mission to help Hurricane Florence victims, he opened up his own wallet so he could contribute over $1,000 to the cause.
With $50 in her pocket, Tench first went into the Walmart in Garner earlier this month with the intention of spending the last of her money on tee-shirts and underwear for a local hurricane relief shelter.
The shelter had told her that while people were able to take hot showers at their facilities, they did not usually have clean clothes that they could change into – so Trench was determined to spend her $50 on clothes.
Upon arriving at the Walmart, she asked the manager, Jeff Jobes, if she could have some sort of discount for the clothing – and she was stunned by his response.
"When Jeff … heard about the plight of the people at the shelter he didn't give me a discount," Trench wrote in an emotional Facebook post. "Instead, he armed me with one of his associates (Alex) and a shopping cart and told her to fill it… on him.
The story does not stop there, however – the next morning, Jobes texted Tench and asked if there was anything else that the shelter needed for the 400 hurricane victims.
By the time Tench pulled up to the Walmart, Jobes and Alex were pulling together piles of donated goods for the shelter.
Walmart as a company has additionally offered to donate $2 for every $1 donated to their Hurricane Florence relief fund, which has already raised over $7.5 million.