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Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.
Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post-transplant transfer of donor-derived, CMV-directed, T cells. Fast and cost efficient methods to generate CMV-specific T cells are, therefore, warranted. The current study utilized peptide-pulsed and adenovirus-transduced dendritic cells (DC) to generate CMV-restricted T cells. After one stimulation with CMV pp65495-503 peptide-pulsed DC and three re-stimulations with peptide-pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide-specific lytic activity. After only one stimulation, pp65495-503-restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000-fold in 2 weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full-length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV-specific CD4+ T-helper cells. This approach would stimulate multiple-epitope populations of pp65-specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full-length antigens may prove to be potent vaccines against viral pathogens and cancer.
BACKGROUND. In order to develop experimental immunotherapy for prostate and breast cancer it is of outmost importance to have representative target cell lines that through human leukocyte antigen (HLA) class I molecules present relevant levels of peptides from tumor-associated antigens for cytotoxic T lymphocyte (CTL) recognition. METHODS. We sequenced the HLA-A and HLA-B loci of eight commonly used prostate and breast cancer cell lines and analyzed the surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD54 by flow cytometry. We also analyzed the cell lines for mRNA expression from 25 genes reported to be specifically or preferentially expressed by prostate cells. RESULTS. Among the analyzed cell lines we found that LNCaP, PC-346C and MCF-7 are HLA-A*0201 positive. However, the HLA-A2 expression level is low and only MCF-7 upregulates HLA-A2 in response to IFN-γ stimulation. MCF-7 also expresses high levels of CD54, which further improve its value as a CTL target cell line. On the other hand, LNCaP and PC-346C express 25 and 23 out of 25 prostate-related genes, respectively, while MCF-7 expresses 16 out of 25 genes. CONCLUSIONS. None of the analyzed prostate cancer cell lines are optimal CTL target cells. However, MCF-7 could in many cases be used as a complement to HLA-A*0201 positive prostate cancer cells. The LNCaP and PC-346C cell lines are rich sources of prostate-related antigens that may be valuable for cancer vaccine development.
BACKGROUND: Expansion of cytotoxic T lymphocytes (CTL) directed against peptide epitopes from antigens that are specifically expressed by normal and neoplastic prostate epithelial cells has during the last years emerged as an interesting therapeutic approach to treat advanced prostate cancer. TCRgamma alternate reading frame protein (TARP) is a protein that in males is specifically expressed by normal prostate epithelial cells and prostate cancer cells. We have evaluated TARP for human leukocyte antigen (HLA)-A*0201-restricted peptides capable of triggering TARP-specific CTL. METHODS: Dendritic cells (DC) were pulsed either with synthetic peptides derived from the natural amino acid sequence of TARP or with cognate peptides having enhanced affinity for HLA-A*0201 due to an N-terminal anchor residue substitution. The peptide-pulsed DC were used to stimulate autologous T cells ex vivo. RESULTS: We were able to generate T cells against TARP(27-35) and TARP(4-13) and their mutated counterparts TARP(V28L)(27-35) and TARP(P5L)(4-13). The use of affinity-enhanced peptides resulted in the generation of T cells recognizing target cells displaying either wild-type or mutated peptide. We further show that TARP-specific T cells can be tetramer-sorted and subsequently expanded to large numbers by general T cell stimulation, with retained specificity and activity. Sorted and expanded T cells, obtained by stimulation with TARP(P5L)(4-13), exert moderate lysis of the TARP-expressing prostate cancer cell line, LNCaP, and breast cancer cell line, MCF-7, indicating that the TARP(4-13) epitope may be endogenously processed and presented by TARP-positive, HLA-A*0201-positive cells. CONCLUSIONS: Our findings suggest that synthetic TARP peptides, such as TARP(P5L)(4-13), may play a role in prostate and breast cancer immunotherapy.
The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor gamma-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.
TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.
TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.
Conditionally replicating adenoviruses are developing as a complement to traditional cancer therapies. Ad[I/PPT-E1A] is an E1B/E3-deleted virus that replicates exclusively in prostate cells, since the expression of E1A is controlled by the recombinant 1.4 kb prostate-specific PPT promoter. The transcriptional integrity of PPT is maintained by the 3.0 kb mouse H19 insulator that was introduced directly upstream of the PPT sequence. In order to increase the cloning capacity to be able to reintroduce E3 sequences in the 35.7 kb Ad[I/PPT-E1A] genome, various shorter insulators were examined in a luciferase reporter gene assay. It was found that the 1.6 kb core H19 insulator (i) improves the activity of PPT, compared to the 3.0 kb full-length insulator, while still maintaining prostate cell specificity and releasing 1.4 kb of space for insertion of additional sequences. To improve the ability of the virus to efficiently lyse infected cells and persist in vivo, we inserted the adenovirus death protein (ADP) or the full-length adenovirus E3 region. The oncolytic activity of PPT-E1A-based viruses was studied using MTS, crystal violet and replication assays. The virus with the reintroduced full-length E3-region (Ad[i/PPT-E1A, E3]) showed the highest cytopathic effects in vitro. Furthermore, this virus suppressed the growth of aggressively growing prostate tumors in vivo. Therefore, we conclude that Ad[i/PPT-E1A, E3] is a prostate-specific oncolytic adenovirus with a high potential for treating localized prostate cancer.
Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37°C for up to 8 hours. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.
The recombinant prostate-specific PPT sequence comprises a prostate-specific antigen enhancer, a PSMA enhancer and a TARP promoter. It is transcriptionally active in human prostate cancer cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer, it has no detectable transcriptional activity. Herein, we describe that the PPT sequence in combination with a two-step transcriptional amplification (TSTA) system becomes active also in murine prostate cancer cells. An adenovirus with TSTA-amplified PPT-controlled expression of the luciferase reporter gene, Ad[PPT/TSTA-Luc], has up to 100-fold higher prostate-specific transcriptional activity than a non-amplified PPT-based adenovirus, Ad[PPT-Luc], in human cells. In addition, Ad[PPT/TSTA-Luc] confers prostate-specific transgene expression in murine cells, with an activity that is approximately 23% of Ad[CMV-Luc] in the transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2 cells. Moreover, to visualize luciferase expression in living mice a charge-coupled device camera was used. Ad[PPT/TSTA-Luc] yielded approximately 30-fold higher transgene expression than Ad[PPT-Luc] in LNCaP tumor xenografts. Importantly, Ad[PPT/TSTA-Luc] also showed activity in murine TRAMP-C2 tumors, whereas Ad[PPT-Luc] activity was undetectable. These results highlight that the recombinant PPT sequence is active in murine prostate cancer cells when augmented by a TSTA system. This finding opens up for preclinical studies with prostate-specific therapeutic gene expression in immunocompetent mice.
BACKGROUND: The interaction between CD40 ligand (CD40L) and CD40 on antigen presenting cells is essential for the initiation of antigen-specific T-cell responses, whereas CD40L stimulation of CD40+ tumor cells can induce cellular apoptosis. We investigated the anti-tumor effects induced by CD40L gene transfer into the mouse prostate adenocarcinoma cell line TRAMP-C2, both in vitro and in vivo.
METHODS: TRAMP-C2 cells were transduced with an adenoviral vector encoding CD40L (AdCD40L). The induced expression of co-stimulatory molecules and cell viability was analyzed. AdCD40L-transduced TRAMP-C2 cells were used in prophylactic vaccination studies, while therapeutic studies were performed using peritumoral injections of AdCD40L.
RESULTS: AdCD40L yielded reduced TRAMP-C2 cell viability and induced apoptosis in vitro. Vaccination with CD40L-expressing TRAMP-C2 cells induced anti-tumor immunity and peritumoral AdCD40L injections induced tumor growth suppression.
CONCLUSIONS: Our observations highlight the therapeutic potential of using AdCD40L as a monotherapy or in combination with conventional chemotherapy or novel therapies (e.g., oncolytic viruses). The use of AdCD40L offers an attractive option for future clinical trials.
Purpose: The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19(+) B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy.
Patients and Methods: Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array.
Results: Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14(+)CD33(+)HLA(-)DR(-)) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2.
Conclusions: Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.
Oncolytic viruses are emerging as anticancer agents, and they have also shown great promise for use against neuroendocrine tumors. Many viruses have a natural tropism for replication in tumor cells. Others can be genetically engineered to selectively kill tumor cells. Viruses have some advantages as therapeutic agents over current cytotoxic drugs and small molecules. They replicate in tumor cells and thereby increase in number over time leading to increased dosage. They are immunogenic and can alter the immunosuppressive tumor microenvironment and activate immune effector cells. They have also been shown to be able to kill drug-resistant cancer stem cells. This article reviews the recent literature on oncolytic viruses used so far for neuroendocrine tumors and indicates important issues to focus on in the future.
T cell immunotherapy is a promising approach to treat disseminated cancer. However, it has been limited by the ability to isolate and expand T cells restricted to tumour-associated antigens. Using ex vivo gene transfer, T cells from patients can be genetically engineered to express a novel T cell receptor or chimeric antigen receptor to specifically recognize a tumour-associated antigen and thereby selectively kill tumour cells. Indeed, genetically engineered T cells have recently been successfully used for cancer treatment in a small number of patients. Here we review the recent progress in the field, and summarize the challenges that lie ahead and the strategies being used to overcome them.
Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.
Autologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens (TAAs) are frequently used as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidence indicate that ex vivo engineered DCs are poor in migration and in fact do not directly present TAA epitopes to naïve T cells in vivo. Instead, it is proposed that bystander host DCs take up material from vaccine-DCs, migrate and subsequently initiate antitumor T-cell responses. We used mouse models to examine the possibility of using pro-inflammatory allogeneic DCs (alloDCs) to activate host DCs and enable them to promote antigen-specific T-cell immunity. We found that alloDCs were able to initiate host DC activation and migration to draining lymph node leading to T-cell activation. The pro-inflammatory milieu created by alloDCs also led to recruitment of NK cells and neutrophils at the site of injection. Vaccination with alloDCs combined with Ad5M(gp100), an infection-enhanced adenovirus encoding the human melanoma-associated antigen gp100 resulted in generation of CD8+ T cells with a T-cell receptor (TCR) specific for the gp10025-33 epitope (gp100-TCR+). Ad5M(gp100)-alloDC vaccination in combination with transfer of gp100-specific pmel-1 T cells resulted in prolonged survival of B16-F10 melanoma-bearing mice and altered the composition of the tumor microenvironment (TME). We hereby propose that alloDCs together with TAA- or neoepitope-encoding Ad5M can become an "off-the-shelf" cancer vaccine, which can reverse the TME-induced immunosuppression and induce host cellular anti-tumor immune responses in patients without the need of a time-consuming preparation step of autologous DCs.
Accumulating evidence support an important role for endogenous bystander dendritic cells (DCs) in the efficiency of autologous patient-derived DC-vaccines, as bystander DCs take up material from vaccine-DCs, migrate to draining lymph node and initiate antitumor T-cell responses. We examined the possibility of using allogeneic DCs as vaccine-DCs to activate bystander immune cells and promote antigen-specific T-cell responses. We demonstrate that human DCs matured with polyI:C, R848 and IFN-γ (denoted COMBIG) in combination with an infection-enhanced adenovirus vector (denoted Ad5M) exhibit a pro-inflammatory state. COMBIG/Ad5M-matured allogeneic DCs (alloDCs) efficiently activated T-cells and NK-cells in allogeneic co-culture experiments. The secretion of immunostimulatory factors during the co-culture promoted the maturation of bystander-DCs, which efficiently cross-presented a model-antigen to activate antigen-specific CD8+ T-cells in vitro. We propose that alloDCs, in combination with Ad5M as loading vehicle, may be a cost-effective and logistically simplified DC vaccination strategy to induce anti-tumor immune responses in cancer patients.
Mesenchymal stromal cells (MSC) have been under investigation for a number of therapies andhave lately been in focus as immunosuppressive actors in the field of transplantation. Herein we haveextended our previously published in vitro model of MSC-islets in an experimental setting of islettransplantation to the abdominal muscle.Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscletissue of NOD-scid ILR2γnull mice and cellular interactions were investigated by confocal microscopy.
The MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, weshow a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-isletscompared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human isletendothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric bloodvessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltratingmacrophages.
Here, in our experimental in vivo model of composite human islets and luciferase-GFP-transducedhuman MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In thismodel of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration.Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naive CD4 cells and demonstrate their efficacy in the EAE model.
CD4+T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.
The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptomfree mice were echallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.
CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.
Neuroendocrine tumors (NETs) consist of heterogeneous neoplasms. The neuroendocrine cells of the human body are confined to certain organs, such as the thyroid, pancreas and adrenals, or they are dispersed throughout the body in the respiratory tract and in the intestinal mucosa. The cells belong to the diffuse endocrine cell system, share a neuroendocrine phenotype, and accumulate precursor molecules which are then processed into hormones, peptides or amines. The tightly controlled release on stimulation is either to the blood stream or adjacent cells or neurons. Neuroendocrine cells regulate various processes in the human body, such as gastrointestinal secretion, blood pressure and response to stress. NETs present a wide spectrum of malignant diseases from rather benign to very malignant and lethal variants. NETs may occur in any organ, but are mainly detected in the gastroenteropancreatic system and in the lungs. The understanding of NET biology and treatments has changed dramatically during the last decade. Today, the main problems that clinicians and translational scientists face in overcoming these malignancies relate to various aspects within the molecular pathogenesis of NETs. This chapter focuses on the importance of novel biomarkers: microRNA and microRNA inhibitors; DNA methylation and epigenetics, and immunotherapy and virotherapy to develop novel treatments for NETs.
Background: Adoptive transfer of genetically engineered autologous T-cells is becoming a successful therapy for cancer. The avidity of the engineered T-cells is of crucial importance for therapy success. We have in the past cloned a T-cell receptor (TCR) that recognizes an HLA-A2 (MHC class I)-restricted peptide from the prostate and breast cancer- associated antigen TARP. Herein we perform a side-by-side comparison of the TARP-specific TCR (TARP-TCR) with a newly cloned TCR specific for an HLA-A2-restricted peptide from the cytomegalovirus (CMV) pp65 antigen. Results: Both CD8(+) T-cells and CD4(+) T-cells transduced with the HLA-A2-restricted TARP-TCR could readily be detected by multimer analysis, indicating that the binding is rather strong, since binding occured also without the CD8 co-receptor of HLA-A2. Not surprisingly, the TARP-TCR, which is directed against a self-antigen, had weaker binding to the HLA-A2/peptide complex than the CMV pp65-specific TCR (pp65-TCR), which is directed against a viral epitope. Higher peptide concentrations were needed to achieve efficient cytokine release and killing of target cells when the TARP- TCR was used. We further introduce the LigandTracer technology to study cell-cell interactions in real time by evaluating the interaction between TCR-engineered T-cells and peptide-pulsed cancer cells. We were able to successfully detect TCR-engineered T-cell binding kinetics to the target cells. We also used the xCELLigence technology to analyzed cell growth of target cells to assess the killing potency of the TCR-engineered T-cells. T-cells transduced with the pp65 - TCR exhibited more pronounced cytotoxicity, being able to kill their targets at both lower effector to target ratios and lower peptide concentrations. Conclusion: The combination of binding assay with functional assays yields data suggesting that TARP- TCR-engineered T-cells bind to their target, but need more antigen stimulation compared to the pp65-TCR to achieve full effector response. Nonetheless, we believe that the TARP- TCR is an attractive candidate for immunotherapy development for prostate and/or breast cancer.
Cancer immunotherapy was selected as the Breakthrough of the Year 2013 by the editors of Science, in part because of the successful treatment of refractory hematological malignancies with adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells. Effective treatment of B cell leukemia may pave the road to future treatment of solid tumors, using similar approaches. The prostate expresses many unique proteins and, since the prostate gland is a dispensable organ, CAR T cells can potentially be used to target these tissue-specific antigens. However, the location and composition of prostate cancer metastases complicate the task of treating these tumors. It is therefore likely that more sophisticated CAR T cell approaches are going to be required for prostate metastasis than for B cell malignancies. Two main challenges that need to be resolved are how to increase the migration and infiltration of CAR T cells into prostate cancer bone metastases and how to counteract the immunosuppressive microenvironment found in bone lesions. Inclusion of homing (chemokine) receptors in CAR T cells may improve their recruitment to bone metastases, as may antibody-based combination therapies to normalize the tumor vasculature. Optimal activation of CAR T cells through the introduction of multiple costimulatory domains would help to overcome inhibitory signals from the tumor microenvironment. Likewise, combination therapy with checkpoint inhibitors that can reduce tumor immunosuppression may help improve efficacy. Other elegant approaches such as induced expression of immune stimulatory cytokines upon target recognition may also help to recruit other effector immune cells to metastatic sites. Although toxicities are difficult to predict in prostate cancer, severe on-target/offtumor toxicities have been observed in clinical trials with use of CAR T cells against hematological malignancies; therefore, the choice of the target antigen is going to be crucial. This review focuses on different means of accomplishing maximal effectiveness of CAR T cell therapy for prostate cancer bone metastases while minimizing side effects and CAR T cell-associated toxicities. CAR T cell-based therapies for prostate cancer have the potential to be a therapy model for other solid tumors.
To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2–restricted T-cell recptor γ-chain alternate reading-frame protein (TARP)4–13 epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2+ prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP4–13 epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.
Adoptive transfer of T cells genetically engineered with a chimeric antigen receptor (CAR) has successfully been used to treat both chronic and acute lymphocytic leukemia as well as other hematological cancers. Experimental therapy with CAR-engineered T cells has also shown promising results on solid tumors. The prostate stem cell antigen (PSCA) is a protein expressed on the surface of prostate epithelial cells as well as in primary and metastatic prostate cancer cells and therefore a promising target for immunotherapy of prostate cancer.
We developed a third-generation CAR against PSCA including the CD28, OX-40 and CD3 zeta signaling domains. T cells were transduced with a lentivirus encoding the PSCA-CAR and evaluated for cytokine production (paired Student's t-test), proliferation (paired Student's t-test), CD107a expression (paired Student's t-test) and target cell killing in vitro and tumor growth and survival in vivo (Log-rank test comparing Kaplan-Meier survival curves).
PSCA-CAR T cells exhibit specific interferon (IFN)-gamma and interleukin (IL)-2 secretion and specific proliferation in response to PSCA-expressing target cells. Furthermore, the PSCA-CAR-engineered T cells efficiently kill PSCA-expressing tumor cells in vitro and systemic treatment with PSCA-CAR-engineered T cells significantly delays subcutaneous tumor growth and prolongs survival of mice.
Our data confirms that PSCA-CAR T cells may be developed for treatment of prostate cancer.
Chimeric antigen receptor (CAR) T-cell therapy is a new successful treatment for refractory B-cell leukemia. Successful therapeutic outcome depends on long-term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV-S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19(+) target cell recognition as LV-engineered T cells and are as effective in controlling tumor growth in vivo We propose that NILV-S/MAR vectors are superior to current options as they enable long-term transgene expression without the risk of insertional mutagenesis and genotoxicity.
Adoptive transfer of patient-derived T-cells engineered with a chimeric antigen receptor (CAR) targeting the pan-B-cell marker CD19 has led to complete remission in patients with B-cell leukemias while response rates are more modest for B-cell lymphomas. This can be attributed to the fact that the semi-solid structure of lymphomas impedes T-cell infiltration and that the immune suppressive microenvironment within these tumors dampens the effect of CAR T-cells. These obstacles are even more pronounced for solid tumors where dense and often highly immunosuppressive structures are found. This article focuses on different aspects of how to improve CAR T-cells for solid tumors, primarily by decreasing their sensitivity to the harsh tumor microenvironment, by altering the immunosuppressive microenvironment inside tumors and by inducing bystander immunity.
Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the "rapid expansion protocol" (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.
Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoterhas not yet been characterized. We identified that a 0.5 kb DNA fragment upstream of the SCG3 gene can selectively drivetransgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased and specificity maintained,by addition of the human achaete‐scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5‐basedadenovirus, where the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cellpenetratingpeptide (Tat‐PTD) in the virus capsid, which we have previously shown results in increased adenovirus transductionefficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1‐SCG3‐E1A), shows selective and efficient killing of neuroblastomacell lines in vitro, including cisplatin‐, etoposide‐ and doxorubicin‐insensitive neuroblastoma cells. Furthermore, it delays tumorgrowth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, wereport a novel oncolytic adenovirus with potential use for neuroblastoma therapy.
B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.
Glioblastoma is an aggressive brain tumor characterized by an abnormal blood vasculature that is hyperpermeable. Here, we report a novel role for CD93 in regulating angiogenesis in this setting by modulating cell-cell and cell-matrix adhesion of endothelial cells. Tissue microarray analysis demonstrated that vascular expression of CD93 was correlated with poor survival in a clinical cohort of patients with high-grade astrocytic glioma. Similarly, intracranial growth in the GL261 mouse model of glioma was delayed significantly in CD93(-/-) hosts, resulting in improved survival compared with wild-type mice. This effect was associated with increased vascular permeability and decreased vascular perfusion of tumors, indicating reduced vessel functionality in the absence of CD93. RNAi-mediated attenuation of CD93 in endothelial cells diminished VEGF-induced tube formation in a three-dimensional collagen gel. CD93 was required for efficient endothelial cell migration and proper cell polarization in vitro. Further, in endothelial cells where CD93 was attenuated, decreased cell spreading led to a severe reduction in cell adhesion, a lack of proper cell contacts, a loss of VE-cadherin, and aberrant actin stress fiber formation. Our results identify CD93 as a key regulator of glioma angiogenesis and vascular function, acting via cytoskeletal rearrangements required for cell-cell and cell-matrix adhesion.
Purpose: The use of replication-selective oncolytic adenoviruses is an emerging therapeutic approach for cancer, which thus far has not been employed for carcinoids. We therefore constructed Ad[CgA-E1A], a novel replication-selective oncolytic adenovirus, where the chromogranin A (CgA) promoter controls expression of the adenoviral E1A gene.
Experimental Design: The Ad[CgA-E1A] virus was evaluated for E1A protein expression, replication ability, and cytolytic activity in various cell lines. It was also evaluated for treatment of xenografted human carcinoid tumors in nude mice. To use Ad[CgA-E1A] for the treatment of carcinoid liver metastases, it is important that normal hepatocytes do not support virus replication to minimize hepatotoxicity. We therefore evaluated CgA protein expression in normal hepatocytes. We also evaluated CgA gene expression in normal hepatocytes and microdissected tumor cells from carcinoid metastases.
Results: We found that Ad[CgA-E1A] replicates similarly to wild-type virus in tumor cells with neuroendocrine features, including the BON carcinoid cell line and the SH-SY-5Y neuroblastoma cell lines, whereas it is attenuated in other cell types. Thus, cells where the CgA promoter is active are selectively killed. We also found that Ad[CgA-E1A] is able to suppress fast-growing human BON carcinoid tumors in nude mice. Furthermore, CgA is highly expressed in microdissected cells from carcinoid metastases, whereas it is not expressed in normal hepatocytes.
Conclusion: Ad[CgA-E1A] is an interesting agent for the treatment of carcinoid liver metastases in conjunction with standard therapy for these malignancies.
We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackie-adenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5 while it transduces normal hepatocytes at about 50% of Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systematic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.
Tumors exhibit immune escape properties that promote their survival. These properties include modulation of Ag presentation, secretion of immunosuppressive factors, resistance to apoptosis, and induction of immune deviation, e.g., shifting from Th1- to Th2-type responses. These escape mechanisms have proven to hamper several immunotherapeutic strategies, and efforts need to be taken to revert this situation. We have studied the immunological effects of introducing CD40 ligand (CD40L), a potent dendritic cell activation molecule, into the tumor micromilieu by adenoviral gene transfer. For this purpose, a murine bladder cancer model (MB49) was used in C57BL/6 mice. The MB49 cells are known to induce IL-10 in the tumor environment. IL-10 potently inhibits the maturation of dendritic cells and thereby also the activation of CTLs. In this paper we show that CD40L immunogene therapy suppresses IL-10 and TGF-beta production (2-fold decrease) and induces a typical Th1-type response in the tumor area (200-fold increase in IL-12 production). The antitumor responses obtained were MB49 cell specific, and the cytotoxicity of the stimulated CD8(+) cells could be blocked by IL-10. Adenovirus CD40L therapy was capable of regressing small tumors (five of six animals were tumor free) and inhibiting the progression of larger tumors even in the presence of other escape mechanisms, such as apoptosis resistance. Furthermore, CD40L-transduced MB49 cells promoted the maturation of dendritic cells (2-fold increase in IL-12) independently of IL-10. Our results argue for using adenovirus CD40L gene transfer, alone or in combination with other modalities, for the treatment of Th2-dominated tumors.
Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is up-regulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates integrin-β1-signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytical cleavage. The CD93-MMRN2 complex was required for activation of integrin-β1, phosphorylation of focal adhesion kinase (FAK) and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of integrin-β1 and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.


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subset: commoncrawl


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Smells and Tastes
By LilyAllena
@LilyAllena (93)
June 21, 2008 8:41pm CST
Do you have a particular smell or taste that takes you back to a place and time in your childhood?
3 responses
@Katherleen (390)
• China
22 Jun 08
One of my biggest one is BBQ Chips,when i was kid ,i used to get them in the summer.
@cream97 (29154)
• United States
22 Jun 08
Strawberry Shortcake cereal. Peanut Butter, Oatmeal cooked on the stove.. Stew Chicken, this woman smelled like this, after cooking it.. Coconut, me and my cousin would pig out of eating it in the fridge.. Cocoa Butter, my mom used to keep a piece in the fridge sliding door.. All these scents take me back to my younger days.. Plus Chanel No. 5 was the perfume I used to wear.. In middle school, I would wear a perfume called Pearls n Lace.. By Avon.. My grandmother used to wear this.. I believe that she still does till this day!


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SoundDiego TV How the Night Howls Became 'The Band With the Hair'
On this week's all-new episode of SoundDiego TV, we chat with locals the Night Howls about hair, blues and Carl's Jr. for our Spotlight segment. See them for free on Dec. 14 at Black Plague Brewing in Oceanside. (Published Sunday, Dec 9, 2018)


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As gilt yields struggle to improve to levels at the start of the month, annuity rates have decreased by as much as 3%.
Providers have reduced annuity rates across the board with Legal & General reducing their rates by 1.5%-3%. This is a significant decrease as Legal & General have been the current market leaders for much of this year.
The last decrease was on 24 July with Canada Life reducing their annuities for the first time since 15 june. Legal & General remain the market leader as all other providers have also reduced their pension annuity rates. Other providers reducing their rates have been Liverpool Victoria by with their smoker annuities lower by 1.5% and Partnership reducing by up to 2.6%.
The reductions have been due to the significant falls in gilt yields and in particular the 15-year gilt yields for which annuity rates are primarily based.
Annuity rates are expected to reduce further
With gilt yields reducing from 2.29% at the beginning of the month they have reduced to 2.12% today, a fall of 17 basis points and as a rough guide this would result in a reduction in annuity rates of about 1.7%. Gilt yields decreased
by 55 basis points in May and annuity rates have not fully reflected this fall so the likely hood is for rates to fall further. For the latest updates see Annuity Rates Review.
This is a difficult time for pensioners retiring now as annuities continue to fall. An open market option can generate a standard annuity that is 20% higher than the current provider and up to 50% higher for impaired annuities. However, as it may take up to four weeks to transfer the funds from the existing provider to the new provider there is a risk that the quote will expire and new lower annuity rates will apply. The lower rate would apply for the lifetime of the pensioner although it is likely the rate will remain higher than they were offered from the current provider as their rates would also have reduced.
Investors doubt ECB commitment
Since the ECB pledged to save the euro markets around the world have rallied and Europe is higher by up to 2.7%. However, investors are beginning to doubt the ability of the ECB to deliver and so the increases are not so significant. Spanish 10-year bonds are much lower than the 7.66% yield attained on 24 July and are now at 6.64% and under the 7% danger level where a bailout would be required, currently expected to be around 300 billion euros for Spain.
The ECB may restart the Securities Markets Programme (SMP) which involves buying bonds from financial institutions and thereby reducing the borrowing cost to countries of issuing bonds. This would reduce the yields on European bonds and if confidence increases could mean investors will move funds from safe havens such as UK government bonds and gilts to Europe. This could decrease the price of gilts and increase the yield which would mean annuity rates rising slightly later in the year. The combined effect of rising pension annuities and equities would significantly improve the income of pensioners at retirement.
15-year gilt yields rise 14 basis points with annuity rates up for the second month
Yields recover from a low as Donald Trump considers limited trade deal with China


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Saturday June 13th Jacquie and I will be leading "Chuckanut Counterclockwise" starting from the Elementary School in Edison on our tandem. 35 miles 1850 feet.
But wait there's more...
If you still have some power left in your legs we will continue onto Samish
Island another 15 miles 315 feet.
We are a solid Brisk (17-20) on the flats and High Moderate on the hills.
Bring tubes and tools to fix what might need fixing. Lunch At Larbee State
Park mile 28.9. Bring your own lunch.
Meet at 9:45 for a prompt 10:00 departure.
Load the routes onto your GPS devices or print out a cue sheet from the
above links.
Ride happy,
John Lindseth
© Cyclists of Greater Seattle. All rights reserved.
P.O. Box 31423 | Seattle, WA 98103


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A guide to healthy eating as you age
Healthy eating used to be as simple as "eat your greens" and "an apple a day keeps the doctor away". If only it were that…
976 Culburra Rd, Culburra Beach
We'd love to show you what's on offer in our IRT community.
Tell us a date and time that works for you and someone will be in touch to confirm your booking.
Join our boutique community of like minded people over the age of 55. Choose from 36 one and two-bedroom villas with private courtyards, parking and on-site security. The delightful bushland setting and well-appointed villas are sure to make you feel at home.
At IRT we make it easy for you to choose a pricing option that's right for you. Select from our four simple pricing options, calculated as a non-refundable, partially-refundable or fully-refundable deposit. Calculate your potential costs now.Calculate price
Do a little or a lot at IRT Culburra Beach. Your Lifestyle Manager will curate a calendar of community activities for you to choose from. Make new friends, find new hobbies or simply relax in your private courtyard. It's your life, your way, at IRT.
Prepare meals and refreshments in your beautifully appointed kitchen with quality appliances. Entertain family and friends at the community BBQ or arrange for IRT home care meal preparation services if your needs change.
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Healthy eating used to be as simple as "eat your greens" and "an apple a day keeps the doctor away". If only it were that…
Kevin Walker's camellia won grand champion at an Illawarra show.
Our aged care centre offers professional personalised care in modern surroundings. Choose from a range of private and shared rooms, or take a break in one of two carer support suites. Our experienced staff are on hand 24/7 ensuring you're comfortable and well cared for.
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Get to know your neighbours and discover new pastimes with your choice of community activities, entertainment and wellbeing programs. Go on community outings or invite your family and friends to join us for special events. Feel included and connected at IRT Culburra Beach.
Enjoy seasonally based meals and refreshments in your care suite or community dining room. IRT Catering are experts in delicious nutritious catering for aged care residents. Be delighted by the dining experience at IRT Culburra Beach.More about IRT Catering
Understanding residential aged care is confusing.
At IRT we make it easy to find aged care that's right for you, or your loved one.
Would you let someone else write your will, deciding what happens to your financial assets when you pass away? No way, you say.And yet, three…
UPDATED 1/04/2020 As a result of the latest announcement from the Australian Government (29/03/2020) urging people over 70 years of age to self-isolate, and limiting…
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26 OCTOBER 2017, SINGAPORE – Snapcart, the real-time offline shopper and consumer insights startup, has announced that it has closed a US$ 10 million series A funding round. The round was led by existing investor Vickers Venture Partners, and includes funding from Social Capital, Kickstart Ventures and Endeavor Catalysts. Existing investors also followed on, including SMDV, Wavemaker Partners and SPH Ventures.
Snapcart gives shoppers cashback for scanning their receipts in exchange for collecting consumer purchase data. This brings brands much closer to a holistic understanding of shoppers than ever before. It works by leveraging on Artificial Intelligence (AI) to retrieve the data from a scanned receipt and translating it into real-time dashboards. This method can replace the time and cost-consuming manual data collection through surveys or interviewing retailers about their customers' buying behavior.
"Snapcart's goal is to empower companies to get valuable insights into customer data to help them allocate their Marketing efforts more efficiently," says Reynazran Royono, Founder and CEO of Snapcart. He adds, "We really believe our product is essential for the modern marketer, as they can react in real time to changes and trends in consumer behavior. This capital allows our company to expand into new markets, improve the current technology and further advance our product."
Snapcart continues to build state of the art technology and introduce new products to further enhance data capturing and help brands to optimize spending.
Dr Jeffrey Chi, Vice Chairman at Vickers Venture Partners expresses, "We are thrilled that Snapcart is truly disrupting the market research industry that has been around for decades but really slow to innovate. Snapcart's innovative and robust technology builds the company's foundation to scale globally. We're really excited that Snapcart is already beginning not just to generate repeat revenues from their customers but are seeing these revenues grow rapidly. This is a testament as to how useful the product is to their customers."
Launched in September 2015, Snapcart previously closed a US$3 million pre-series A round in 2017. Snapcart currently operates in two countries – Indonesia and Philippines – and recently opened a Singapore office to focus on business development and client acquisition. The app has over 700.000 registered users and is collaborating with over 75 FMCG brands, e.g. Unilever, L'Oreal and Nestlé.
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Pricing a product properly can result in a big difference between success and failure for a company. However, determining the correct price is not an easy fix, read more →
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On Thursday 20th April 2017, "Becoming a Data-driven Organization: Trends and Challenges" event was held in Fairmont Hotel, Senayan. read more →
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The guys at Smythson are getting it all so right, right now. We are quite critical of this brand as we have seen some dreadful schemes, particularly for a brand that sells very expensive, and we are sure worth every penny, merchandise. But, to entice us in? Well, it really wasn't happening. However, someone here has clearly now got their finger on the pulse and tapping into some of the key trends. We adored the Jubilee scheme and this one with their links to jellyfishlamp.com just looks so good. We're seeing references to the 'shanty' trend all around town right now; these things are so exciting to follow and to see how everyone is interpreting them is wonderful. Fantastic guys, another little winner for the high street.
We will continue bringing you the latest in windows as much as we can over the coming weeks
Tuesday, 12 June 2012
Smythson, London
Labels: Smythson
Location: London London, UK
No comments:
Post a Comment
Many thanks for leaving your comments which are always most welcome; we are unable to respond to every single one individually but we do read them all.
(A huge apology for this horrible word verification also which is in place to eliminate the dreaded spam).
Kind Regards
Jonathan & Team
Subscribe to: Post Comments (Atom)


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Directions For ENGN: As a dietary supplement, take 1 scoop with 8 oz (236 mL) of cold water 15-30 minutes before training. To avoid sleeplessness, do not take within 4 hours of sleep.
Warnings: Not for use by those with pre-existing medical conditions; those taking any medications; those under the age of 18; those that are caffeine, niacin, or beta-alanine sensitive or women that are contemplating pregnancy, pregnant or nursing. Consult your physician prior to using this product if you are taking any prescription or over-the-counter medication or supplements. Do not use this product if you are at risk or are being treated for any medical condition including, but not limited to: high or low blood pressure; cardiac arrhythmia; stroke; heart, liver, kidney or thyroid disease; seizure disorder; psychiatric disease; diabetes; difficulty urinating due to prostate enlargement or if you are taking a MAO inhibitor. Discontinue use and consult your healthcare professional if you experience any adverse reaction to this product. Keep out of reach of all children. Do not exceed recommended serving size or suggested use for any reason. Discontinue use 2 weeks prior to surgery.
|* These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure or prevent any disease.


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[Mikuni Pass in Kamishihoro] Red Bridge through Primeval Forest with Fall Foliage Is Picture-perfect
October 23rd, 2017
Mikuni Pass
Mikuni Pass (三国峠) in Kamishihoro located in the north part of Obihiro is the highest pass at 1,139 m above sea level in Hokkaido, and is on National Route 273 (Nukabira National Route (糠平国道)) that connects Kamikawa (上川) with Tokachi (十勝), and penetrates Daisetsuzan National Park (大雪山国立公園) in the north-south direction. There is no house around the Mikuni Pass, a sea of trees such as Sakhalin Fir (トドマツ) and Yezo Spruce (エゾマツ) spreads over. The sea of the trees is covered with fall foliage, so a lot of tourists come to a good place to see the fall foliage. The best time to see the fall foliage is from mid September to early October. Mikuni-toge Lookout (三国峠展望台) is in front of Mikuni Tunnel (三国トンネル). Admission free. A parking lot is free of charge. Mikuni Cafe (三国Cafe), a drinking fountain, a public restroom and the parking lot are provided on the site. The Mikuni Cafe serves meals from late April to late October, and the business hour is from 08:00 to 17:30. Horoka Onsen (幌加温泉) and Nukabira Onsen (糠平温泉) are a little away from here.
Lake Nukabira
Lake Nukabira (糠平湖) in Kamishihoro town located in the north part of Obihiro city is the 2nd largest man-made lake after Lake Shumarinai (朱鞠内湖) in Hokkaido. In the Eastern Daisetsu Area (東大雪エリア), Daisetsuzan National Park, the lake was born by completion of Nukabira Dam (糠平ダム) in 1956. The perimeter of the lake is about 34 km. The maximum water depth is 75 m. The Nukabira means a human-shaped rock in Ainu language. When the Nukabira Dam was completed in 1955, the rock has been flooded in the lake. Nukabira Hot-spring Resort (糠平温泉) is in the southwest side of the Lake Nukabira. Higashi-taisetsu Nature Center (東大雪自然館) is built in the hot-spring resort, and visitors can gather information about the Lake Nukabira. In addition, Horoka Onsen (幌加温泉) is near the Lake Nukabira. Speaking of the Lake Nukabira, old bridges are famous. About 13 bridges such as Taushubetsu River Bridge (タウシュベツ川橋梁) used as former railroad track bridge still have been built, and it becomes popular tourist attractions because a state that is in harmony with landscapes is beautiful. Kamishihoro Town Rail Museum (上士幌町鉄道資料館) is built near the hot spring resort, too. Article is here
Horoka Onsen
Horoka Onsen (幌加温泉) is a hot spring place in Kamishihoro town located in the northern part of Obihiro city. A blue signboard of the Horoka Onsen is a mark along Nukabira National Route (糠平国道 * National Route 273), and visitors turn and go straight for around 1.5 km, and the end of the road is the Horoka Onsen. Only Kanoya Onsen Ryokan (鹿の谷温泉旅館) is built at around 700 m above sea level at the foot of Mt. Horoka in the Higashi-Daisetsu region, Daisetsuzan Natioanl Park (大雪山国立公園 東大雪地区), and is well-known as a secret onsen. It is out of signal area. There is a trailhead for Mt. Nipesotsu, but restraunts, cafes, souvenir shops and convenience stores are not built around here. Lake Nukabira (糠平湖) is near here, and there are Concrete Arches of Former Shihorosen Railway (旧国鉄士幌線廃線跡コンクリートアーチ橋梁群) including Taushubetsugawa Bridge (タウシュベツ川橋梁), and Nukabira Onsen, Nukabira Dam, Forest Truck Railway Eco Rail (森のトロッコ・エコレール), Mikuni Pass (三国峠) and Naitai Highland Farm (ナイタイ高原牧場) are relatively near here, too. Article is here
Nukabira Onsen
Nukabira Onsen (糠平温泉) is a hot-spring place at the south side of Lake Nukabira in Kamishihoro town located in the northern part of Obihiro city. The hot-spring place is formed along Nukabira National Route (National Route 273), and is a base for sightseeing of Higashi-Daisetsu region, Daisetsuzan Natioanl Park (大雪山国立公園 東大雪地区), and there are 9 hot spring accommodations, restaurants, a shop, souvenir shops, footbaths, handbaths, Nukabiragensenkyo Ski Resort (ぬかびら源泉郷スキー場), Higashi-Daisetsu Nature Building (東大雪自然館), Kamishihoro Railway Museum (上士幌鉄道資料館), Higashi-Daisetsu Nature Guide Center (ひがし大雪自然ガイドセンター), Nukabira Campsite (国設ぬかびらキャンプ場). Unfortunately, there is not a convenience store. There are Concrete Arches of Former Shihorosen Railway (旧国鉄士幌線廃線跡コンクリートアーチ橋梁群) including Taushubetsu River Bridge (タウシュベツ川橋梁), Nukabira Dam, Forest Truck Railway Eco Rail (森のトロッコ・エコレール), Mikuni Pass (三国峠) and Naitai Highland Farm (ナイタイ高原牧場) around the Nukabira Onsen. Article is here
Daisetsuzan National Park
Daisetsuzan National Park (大雪山国立公園) in the center of Hokkaido is the largest national park in Japan, the size of 226,764 ha, and has been designed as the national park since 1934, and is the oldest national park in the Hokkaido along with Akan Mashu National Park (阿寒摩周国立公園). A mountain named Daisetsuzan does not exist, and the whole area including the mountains with over 2,000 m above sea level such as Mt. Asahidake (旭岳), the highest mountain in Hokkaido, Mt. Kurodake (黒岳), Mt. Aibestudake (愛別岳), Mt. Tomuraushi (トムラウシ山) is collectively called "Daisetsuzan", and those are called "A roof of the Hokkaido" ,too. Rare nature such as active volcanoes, Lare Columnar joints, a sea of trees and permafrost even in the Hokkaido can be seen. About 250 types of alpine plants grow naturally, and various forms of life such as brown bear, blakiston's fish owl, dolly varden and pine grosbeak inhabit here. By the way, A right notation is "Daisetsuzan", but local people often call "Taisetsuzan". Article is here
Fall Foliage from Mikuni Pass
Visitors can enjoy a primeval forest with fall foliage from the Mikuni-toge Lookout, But Matsumi-ohashi Bridge (松見大橋), about 30 m heigh and about 330 m total length, through the primeval forest from Ryokushin Bridge (緑深橋) is most popular. A lot of the tourists come to take a photo of a combination of curved red bridge and the fall foliage. Mountain range and the sea of the trees with the fall foliage from the Ryokushin Bridge is good, too. The visitors must park their cars on the shoulder of the road because there is not a parking lot. The road has lots of traffic, so Watch out for cars!
|Admission fee
|Free of charge
|Parking lot
|Free parking is provided (15 cars)
|Business hour
|24 hours
|Regular closing day
|Open all year round
|Telephone number
|Home page
|Home page is here
|743 286 332*42
List of Accommodations
Refer to following websites for the accommodations around Mikuni Pass. You can get information regarding online reviews and photos.
Horoka Onsen
|Kanoya Onsen Ryokan(鹿の谷温泉旅館)
|Fax: +81-1564-4-2163
Nukabira Onsen
|Ganso Yumotokan(元祖 湯元館)
|Nukabira Onsen Hotel(糠平温泉ホテル)
|Nukabirakan Kanko Hotel(糠平館観光ホテル)
|Higashitaisetsu Nukabira Youth Hostel(東大雪ぬかびら ユースホステル)
|Onsen Pension Mori-no-Fukuro(ペンション 森のふくろう)
A page of Daisetsuzan National Park is here
Daisetsuzan National Park


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These crochet winter hats are perfect for keeping you and your family during the cold winter months. Some of these hat patterns are available in all sizes, while others can easily be modified for a custom fit.
In this collection you will find several cozy winter hats designed with women in mind, but many can be used for men as well. So here we go…
Crystal Drops Beanie by Crochet For You
Here is a simple beanie with a pretty laced edge for an extra touch of love. The hat pattern is available in all sizes from child to adult men.
Cuddly Soft Slouchy Hat by Underground Crafter
This hat is worked up in a bulky weight yarn, so it not only works up quick, but it'll be super cozy as well. And it's available in all sizes, making it a great go-to pattern for the family.
Half Double Crochet Basic Beanie by Oombawka Design
Here is another hat that is available in all sizes. This one is crocheted in a basic half double crochet, making it a great beginner-friendly pattern.
Dashes Beanie by CrochetKim
This beanie pattern is available in one size only. It features a beautiful texture and color design, which makes it easy match to any outfit.
Beaded Stripes Slouchy Hat by CrochetN'Crafts
Easy to crochet to any size, this pretty textured hat also makes a nice stashbuster pattern.
Simple Shells Slouchy Beanie by Beatrice Ryan Designs
Shells are quick and easy to crochet. And with the pretty colors, this hat is sure to please everyone from the person who crochets to the person who gets to wear it.
Slanted Shell Beanie by EyeLoveKnots
Here is another shelled hat, but this one has a pretty slant to it.
Lolly-Poms Easy Ribbed Beanie by One Dog Woof
Not only does this hat feature a gorgeous knit look, but it's also easy to crochet and easy to adjust to any size from baby to adult.
The Frolic Hat by CrochetN'Crafts
This hat has a nicely textured edge with a pretty, yet simple stitch for the rest of the hat. And best of all, it's easy to crochet to any size.
The Echo Ridge Slouch by My Hobby is Crochet
The self-striping yarn and the beautiful ridged look makes this a design you'll want to crochet for all your friends and family. It is available in one size to fit the average woman.
Swirling Pebbles by ELK Studio
The texture of this hat is crocheted with very basic stitches. But it's beautiful and perfect for an elegant look.
The Marion Slouch by Little Monkeys Crochet
This classy slouch is the perfect accessory to compliment your best outfits. It features a number of stitches to create a beautiful textured design. And it looks like it's a comfortable fit as well.
Moray Slouchy Hat by AG Handmades
I love the spiral design on this beautiful slouch. It's an easy pattern, and can be crocheted to any size you need.
Squish Slouch Hat by Moogly
Crocheted in chunky yarn, this hat works up quick. It is available in one adult size, and comes with several links to helpful videos on the stitches used to crochet this.
Any Size Hat by CrochetN'Crafts
Crochet this pretty textured hat to any size that you like. It has a nice bobbled edge in a contrast color for a super cool look.
If you enjoyed this roundup of crochet winter hats, then you might also enjoy the collection happening over at CPB: Crochet Hats.


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This Workshop explore the basics principles of N.L.P which give students a powerful set of skills and ways of thinking to develop the art of 'self-mastery'. It is usually run in a four hour block and the primary focus is to examine the fundemental aspects of self-mastery which includes the following key areas:-
Understand how we all filter information differently and why this is important to know
Learn how to connect and build deep rapport with others
Learn how to move from 'limiting thoughts' to 'empowering beliefs'
How to lead a 'Value-driven' life.
Learn how to 'respond' rather than 'react'
(Workbooks and notes are included)


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Discussion in 'Map Factory' started by Wilson, Feb 20, 2012.
Old map, new thread. (And so on)
Give feedback or give me random praise. (And so on)
That is all.
Updated to Stage 1 Alpha 6.
Still trying to figure out how that freight container managed to get under there. (third screenshot)
two forklifts carried it.
"To you!" "To me!"
Then i forgot to update thread.
Stage 1 has been Alpha 7 a while now.
Separate names with a comma.


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Sat, Jun 23, 2:17am by Ethan Anderson
Last Updated Mon, Oct 7, 11:41pm
A Lively Mexican Themed Slot with Multiple Reels in the Bonus Game
More Chili is a bright, lively slot full of clichés about Mexico. You'll find a classic Mexican on the reels, complete with hat and moustache. There is also a cart full of chilis, a chicken and a strange blue donkey – which I'll assume represents a piñata.
This is another online pokie game from the Aristocrat back catalogue. It does show signs of age with the design – though remains an entertaining pokie. You can enjoy it online or in live settings.
Other than the classic Aristocrat style of play, there are some pretty good features that are included in the More Chilli pokies. Here's the top 3 list:
400+ Games
1-2 Days
+1 More
Before you spin, you can choose to play 5 extra coins on top of the 25 you'll play to cover the win-lines. This makes a difference to the free spins bonus. Instead of the usual 12, you'll get 15. As you will see, this gives you more time to collect chilis, earning those extra sets of reels.
You need 3 of the special bonus symbols to start this feature, these show money bags.
Next, you'll see a short cartoon animation, where a Mexican guy appears on the reels and shouts 'more Chilli!'. You then see two separate reels, which spin at the same time. Wins on either count towards your total – and the bonus can be retriggered on either.
During this feature, red chili peppers (which also appear in the base game) have a special function. If you collect 9 of them anywhere on either set of reels, you'll earn a whole new reel set. If you then collect 9 more, you earn a 4thand final reel set.
Wins from all 4 can accumulate fast – and you can retrigger this bonus on any of those sets.
Like many of the older Aristocrat pokies, More Chilli uses win-lines instead of the reel-power system. There are 25 win-lines crossing a regular 5×3 grid. You can bet from 1c per line and up. If you add the extra 5 coins, you'll not only get more free spins – the bonus feature can be triggered at random at the end of any spin too. If you don't want to play with real-money, thats ok! Most of Aristocrat's pokies are available to play for free, including More Chilli! Scroll up the page to play for free.
The More Chilli winning symbols are cartoon-like and tongue in cheek. The highest paying symbol is also the wild. This is a Mexican guy, with patterned hat and jet-black moustache. He is smiling and will jump for joy when involved in a win. As the wild he will substitute for the regular symbols, though not the money bag bonus symbols or the special red chilis. Line up 5 of these wilds on a win-line, and 2000 coins are coming your way.
Next comes the strange looking blue creature, worth 1000 coins for 5 of a kind. Like the wild, this symbol gives prizes for just 2 from the left – the other symbols need a minimum of 3.
Unique symbols are completed by a chicken, cart full of chilis, blazing sun and a bottle of tequila. Playing card filler symbols make up the smaller and more regular wins.
Above the reels of this free online pokie, you'll see a dusk sky, with bright red rocky mountains on either side. The backing of the reels is a plain beige, which highlights the brightly coloured symbols nicely. The farmer guy is stands out, grinning at you from the reels and jumping in an old-style animation when involved in winning combinations. The red chili symbols don't play a role in the base game – though they certainly look good enough to eat.
Just when you think this machine is plain, the bonus feature brings it to life. You'll get a full-size Mexican introducing the feature, and a monster giving you your win total at the end. The setting of the bonus game is a town, you can see the rooftops and country beyond.
There is also a Mexican guitar tune playing in the feature, which contrasts with the standard Aristocrat pokies beeps and clicks. When you collect chilis, an animation sends them to the top of the screen. The bonus reels are small, though I'm sure those extra wins will more than make up for their size!
Another classic poker machine from Aristocrat. This game is fun, lively and certainly has the potential for big wins. The base game is ordinary, though you'll find plenty of extra wins with the wild symbols. It comes to life in the bonus game. Here you start with 2 sets of reels – and collect chilli peppers to earn up to two more.


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TERRE HAUTE, Ind. (WTHI) - Indiana State University has named the two finalists in the Presidential search.
The two finalists are Deborah Curtis, provost and chief learning officer at the University of Central Missouri, and Michael Licari, who has served as provost and vice president for academic affairs at Indiana State since July 2015.
"We are very pleased to have two incredibly strong candidates for this critically important position, both of whom understand and fully support Indiana State's mission and both of whom we feel have the skills and experience to be successful as its next president. We were prepared for the possibility of three or four finalists, but in the end these two candidates distinguished themselves among all others," said Jeff Taylor, chair of the presidential search committee and secretary of the ISU Board of Trustees.
70 candidates were in the beginning pool of this search and the search committee narrowed it down to nine before selecting the two finalists.
Curtis earned her Ph.D. from Indiana State and also taught at the former University School. In addition to serving as provost of Indiana State, Licari has a son who is a freshman at the university.
Licari's interview will take place on Oct. 25-26, while Curtis will visit campus on Nov. 1-2.
In addition to a series of private interview sessions with the trustees and other campus groups, each candidate will have a dessert reception with community and volunteer leaders and open sessions for staff, students and faculty. The schedules for these sessions are:
Oct. 25, 2017 (Dr. Michael Licari)
1:15 - 2:45 p.m. Dessert reception with community and volunteer leaders
Magna Carta Room, Federal Hall (Scott College of Business)
3 - 4 p.m. Candidate presentation and open session for faculty
Heritage Ballroom, Tirey Hall
Oct. 26, 2017 (Dr. Michael Licari)
3 - 4 p.m. Open session for staff
Heritage Ballroom, Tirey Hall
4 - 5 p.m. Open session for students
Heritage Ballroom, Tirey Hall
Nov. 1, 2017 (Dr. Deborah Curtis)
1:15 - 2:45 p.m. Dessert reception with community and volunteer leaders
Heritage Lounge, Tirey Hall
3 - 4 p.m. Open session for staff
Heritage Ballroom, Tirey Hall
4 - 5 p.m. Open session for students
Heritage Ballroom, Tirey Hall
Nov. 2, 2017 (Dr. Deborah Curtis)
3 - 4 p.m. Candidate presentation and open session for faculty
Heritage Ballroom, Tirey Hall
The Board of Trustees anticipates naming a new president before Thanksgiving.
Related Content
- Indiana State University announces two finalists in Presidential search
- Committee hosts university presidential candidate
- "...really a no brainer..." News 10 sits down with finalist in ISU Presidential search
- Board of Trustees announce 12th President of Indiana State University
- Indiana State University announces minimum wage increase for student workers
- Indiana State University announces completion date for Hulman Center renovation
- Indiana State University projects attract 1,200 jobs
- Indiana State University receives $64 million project
- Indiana State University hosts student job fair
- Indiana State University helping displaced students


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Expat Meme
I found this meme on Avoiding Crisis: 210 Days of Self-Exploration.
Name five things you love in your new country
- My Friends
- No coffee to go
- More cultural events than you can wave a stick at
- How technology hasn't completely isolated people
- Late nights
Name four things you miss from your native country
- My family and friends
- My sense of balance and stablity
- My ability to communicate
- Neighborhood restaurants with spicy "international" food (mexican, thai, indian, etc.)
Name three things that annoy you in your new country
- No bike lanes
- Relative expense of technology
- The garantia system of renting apartments
Name two things that surprise you (or surprised you when you arrived) in your new country
- Everyone has little dogs
- The fashion
Name one thing you would miss in your new country if you had to leave
- Kissing on the cheek


text: Gary Clancy - October 28, 2010 The Chase Phase Each Fall Whitetail Bucks Engage In Brief But Foolish Games Of Pursuit By Gary Clancy Having worked themselves into a frenzy making scrapes, but with the breeding portion of the rut still well away, bucks begin pressing does. This brief period during the fall results in some incredibly intense deer movement when buck sightings increase dramatically.Back in my high school days I played a little third base. There was a skinny kid from one of the neighboring schools who gave me fits. The kid batted right-handed and was a straight pull hitter. I mean, this kid had never hit a ball to right in his life. The kid would step up to the plate, dig in, wave the lumber and then cock his head a bit to stare at me over his left shoulder. If I shifted toward the bag, the skinny kid would slap one through the gap between me and the shortstop. The next time the kid came up I would make sure I covered that hole and the kid would drill one right over the bag. Every time we played that team, the skinny kid made me look bad.I got to thinking about those old high school games and the skinny kid after an exciting, but frustrating, day of bowhunting in northeast Iowa. It was the second week of November, the 10th to be precise. The morning was one of those you dream about--clear, cold and so calm you could hear a field mouse expel gas at 100 yards. It was one of those mornings when you can just feel it, know what I mean? Something good was going to happen on this day. Twice in the 20 minutes it took for shooting light to seep out of the night sky I heard deer running in the timber below the ridge on which I perched. It was so still that the deer sounded like they were right under the wind-twisted oak to which I had strapped my stand, but they were actually far below along the tired, little creek. The light was still murky when I heard another deer coming my way. The doe blasted from the edge of the woods, darted into a field of set aside and came running full-tilt right at my tree along the seam where frost-coated grass met dark timber. Right behind her came a hog-fat, heavy-horned, wide-shouldered Iowa mega-buck. In an instant the doe blew right past my tree, with the buck chugging along 20 yards behind. I drew and grunted at the buck with my mouth to get him to stop. If he heard me, he sure didn't acknowledge it. I let out a loud blaaat! He kept right on motoring. Flustered now, I swung with him, hit the release and watched the yellow and white fletching disappear into the grass six feet behind his fat rear end. I was still kicking myself for taking a shot I know better than to take when another doe came running full-tilt over a gentle hill in the open field. Behind her were a pair of immature bucks, both looking to be basket-racked eight-points. The trio darted into a finger of brush jutting into the field about 200 yards south of my perch and then disappeared. The next hour was quiet.Soon I heard deer running again, this time behind me. It was another doe being chased by a buck. They ran below my stand in the heavy timber and then came squirting out that same finger of brush the threesome had run into an hour earlier. Through binoculars I studied the buck as he hot-footed after the doe across the field. Not huge, but a nice buck, long tines but lacking the mass of a mature animal. I started to think about moving my stand to that brushy draw. An hour later, having seen no more deer, I did just that. I planned to hunt all day; might as well be in the best spot, right? Right! Guess where the next deer I saw walked? Yup, right under that oak I had just vacated. Nice buck too and walking, not running. By dark I had seen three more bucks, although I think two of them were the same eight-points I had seen cross the field earlier. One of them ran a doe in circles around that oak in which I had spent most of the morning. Kind of reminded me of how I felt when that skinny kid kept slapping that ball through the holes where I used to be. That's the chase phase for you. Lots of action, but trying to pick the precise stand location to best take advantage of that activity will drive you nuts.During most of the rut the author is partial to using buck decoys to lure whitetails; however, during the chase phase he finds that a doe decoy gets better results. He likes to make sure the decoy is easily visible from a number of directions.WHY THEY CHASE There are two main periods to the annual whitetail rut--the scraping period and the breeding period. In the northern half of the whitetail's range, these two periods are well-defined, because the breeding period occurs at a predictable time each year and is short and intense in duration. This is nature's way of ensuring that the fawns will be dropped late enough in the spring to miss those deadly spring blizzards, but not so late that the youngsters are too small to make it through their first winter. In the southern states, where it is not critical that does give birth during a short and specific period, the breeding tends to be drug out over weeks and in some cases months, making it impossible to predict the timing of the main periods of the rut, much less the chase phase.In those areas with a well-defined rut, the chase phase occurs at the tag end of the scraping period and just prior to the first wave of does entering estrus. The bucks have worked themselves into a frenzy by this time. They are primed and ready for action. To ensure that a doe has a buck already on the hook when she is ready to be bred, does begin to give off scent signals a day or two prior to actually entering estrus. These signals are picked up by the bucks, and the chase is on. Of course the doe is not ready to stand for the buck yet, so the doe has to stay on the move constantly. When you get multiple does in this condition in your hunting area, you are in for a real circus. When it comes to seeing numbers of deer, both bucks and does, no other time compares with the short, but intense chase phase of the rut.SETTING A DATE Because the chase phase is short-lived, timing it is critical. Fortunately, predicting when the chase phase will occur is not difficult. The peak of the breeding period, which is a five-to-seven-day-long whitetail orgy during which about 70 percent of the does are bred, falls on about the same dates annually. Granted, the moon phase, the weather, herd condition and hunting pressure can all have an influence on the exact timing and the intensity of daytime movement during the breeding period, but the dates will not vary by more than a few days in either direction from year to year. Nearly all game and fish departments will be able to furnish you with the dates of the peak of the breeding period in the state you are hunting.Once you know that the peak of the breeding period is from, let's say, November 15-20 in the state you are hunting, you can bet that the chase phase is going to occur sometime in the week prior to the beginning of the peak of breeding. In this case I would be looking for chase phase action between November 8-15. Don't expect the chase phase to last the entire eight days, however. A couple of days of nonstop chasing is more the norm, but the chase phase will occur sometime during this time period; the exact timing depending upon the dates of the peak of breeding for that year.BOWHUNTING TACTICS The chase phase is one of my favorite times to be in the woods. If you are in decent whitetail country, seeing deer is almost a sure thing. However, seeing deer is the easy part. Setting up so that one of those bucks saunters by within bow range is the frustrating part of hunting the chase phase. Whitetail deer are unpredictable critters most of the time anyway, but when you've got a bunch of sex-crazed bucks chasing unwilling, frightened does around, the unpredictability factor skyrockets. I don't have any sure cures, but here are some of the things that have worked for me.The results of a successful hunt during the chase period. If you can hunt all day at this time, by all means do so. It's a certainty the bucks will be doing the same.Use a doe decoy. I do a lot of hunting over decoys and usually I prefer a buck decoy, but during the chase phase, because bucks tend to rush up to every doe they see, I've found that a doe decoy or two draws more attention than a buck decoy. Be sure the doe decoy is easily visible. Field edges, clear-cuts, logging roads, natural woodland openings and tree-studded pastures are all good bets. The more directions from which a buck can spot the decoy and the greater the distance, the more bucks your decoy will attract.Use a good doe-in-estrus urine. Don't put it right on the decoy, however. If you do, the odor will get on your clothing when you handle the decoy; and you don't want to be smelling like a doe-in-heat and draw attention to yourself. I like to jab a stick in the ground at the rear of the decoy and place a cotton ball or scent wick saturated with scent on the stick. Some guys just pour the scent on the ground, but I think the cotton ball or wick holds the scent longer. You don't need the urine to attract the buck to the decoy, but the urine will hold the buck's attention for a long time and allow you to get the perfect angle for the shot.Hunt funnels. That's worn advice, I know, but it is still valid. A buck may wander through the funnel in his search for does, or a harassed doe may pull a buck through the funnel behind her. Either way, you are a winner.Hunt food sources. If you know where the does are doing most of their feeding, that is the place to be when the chase phase is in progress. The bucks know these places too; and although they could care less about eating, they will cruise through these whitetail cafeterias hoping to catch a doe or two chowing down. Great places for a decoy too.Do a lot of calling and rattling. Right now is prime time to grunt in or rattle up a buck. The bucks are going bonkers, and it does not take much to convince them that they are missing out on some hot action when they hear what they think is another buck giving the tending grunt or two bucks fighting.If you can, hunt all day. Sure, the action is most intense morning and evening, but bucks are cruising for does all day long during this period. If you are going to take a break, do it between about 1:30 and an hour before sunset; in my experience this is the period of least movement. I suppose the deer have to rest sometime, even during the chase phase. GET THE NEWSLETTER Join the List and Never Miss a Thing. Sign Me Up


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Metallic Slip-On Shoes
Out of stock
Or 4 payments of More Info
A crackled metallic finish makes these leather slip-on shoes a showstopper wherever you go this season.
- Leather upper
- Cotton and leather lining
- Leather and polyurethane insock
- Polyurethane outsole
- All-over textured metallic finish
- Tommy Hilfiger logo on insock
- Tommy Hilfiger flag plaque on side
Shape & Fit
- Shaft height 6cm
Composition & Care
- 100% leather
Style #
Shipping & Returns
- Free shipping within Australia on all orders of $100 or more
- Simple and easy returns.
- We do not offer exchanges, only refunds. Due to hygiene reasons, earrings, hair pieces, hosiery, underwear bottoms and swim products are unable to be returned for a change of mind or incorrect sized purchases.
- Visit our customer service page for more information regarding payment methods or contact our customer service team for further questions.


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The Guantanamo Bay detention center recently lost its fraternity charter after more than a decade of revelations that it has been abusing members during the pledge process. Brad Beemer, spokesperson for the GTMO fraternity's national organization, described the decision to revoke the charter as unfortunate and avoidable. When asked why they didn't revoke the charter after the 9th pledge had died in 2012, he responded, "Look, a few guys may not be able to take it and die during the pledge process. When the chapter lost its 9th guy, sure, we thought it might've been due to negligence, poor transparency, and a lack of oversight. But we didn't want to jump to any conclusions."
As time went on, however, Guantanamo continued to break rules such as waterboarding and beating pledges prior to giving them their fraternity pin – a strict violation of the North-American Interfraternity Conference's (NIC's) code. Things then escalated with detainee Adnan Latif, who was held for more than 10 years without charges. Besides violating the Geneva Convention, this was also in direct opposition to NIC rules which allow pledges to know why they are being held and earn their escape from confinement by either drinking a 750mL handle of vodka or sorting 1 gallon of sprinkles by color in under 2 hours. It turned out that Guantanamo's torture rooms didn't even have sprinkles in them! How appalling.
But it wasn't until the annual NIC picnic that things reached a boiling point. For their fraternity's skit, the chapter hogtied inmates in the nude on stage and tied others to leashes, forced them to make animal noises, and perform dog tricks. This after the NIC explicitly banned interpretative dance routines for the event. Finally enough was enough.
The fraternity, absolutely disgraced by its chapter in front of the whole conference, decided to strip Guantanamo of its charter.
A 7-year pledge was furious with the decision especially since he was only a few years away from becoming a brother. "Now this place will be swarming with non-Greek losers. Look there's one now!"
The pledge pointed to another inmate wearing cargo shorts and an oversized Led Zeppelin t-shirt. "No way man," responded the inmate in cargo shorts, later identified as Sami Abdul Aziz Salim Allaithy. "I've been here for years."
"Yeah, but you're not one of us. You're not even an enemy combatant!"
"So what?" said Allaithy. "They said I could live here against my will if I wanted. Or not. Then they beat me. Anyways, I earned my greek letters."
Despite the outcry from Guantanamo's members, however, not everyone is upset with the decision. We spoke to a neighbor who was ecstatic that the detention center might finally quiet down. "Finally, we'll have some peace and quiet around here. All those assholes ever did was blast loud music 24/7, it's like they never slept."


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Trojan Bareskin Condoms
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