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Research article | Open | Published: 21 April 2015
Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model
Michael D Roth ORCID: orcid.org/0000-0003-2194-65781 &
Airi Harui1
The complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and the systemic immune system are likely to define critical factors in the host response to cancer. While conventional animal models have identified an array of potential anti-tumor therapies, mouse models often fail to translate into effective human treatments. Our goal is to establish a humanized tumor model as a more effective pre-clinical platform for understanding and manipulating TIL.
The immune system in NOD/SCID/IL-2Rγnull (NSG) mice was reconstituted by the co-administration of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells (PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating cytokines and chemokines were also assessed.
A tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG animals closely matched the features of TIL recovered from primary human prostate cancers. Human cytokines were readily detectible in the serum and exhibited a different profile in animals implanted with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but could not eliminate tumor growth and this effect required the presence of CD4+ T cell help.
Simultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that recapitulates the development of human TIL and allows an assessment of tumor and immune system interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual features and cell populations provides an opportunity for hypothesis testing and outcome monitoring in a humanized system that may be more relevant than conventional mouse models.
Tumor infiltrating lymphocytes (TIL) are a universal feature of human cancers and may hold an important key to unlocking effective cancer immunotherapy [1-3]. TIL exhibit tumor-specificity but are obviously impaired in their capacity to eradicate tumor targets in situ within the tumor microenvironment. While a basic understanding of the composition and phenotype of TIL has come from the study of human tumors, these studies are limited by patient heterogeneity, a lack of access to corresponding lymphoid tissue, and an inability to directly investigate mechanisms and interactions. As a result, researchers have turned to animal models to evaluate mechanisms and therapeutic outcomes [4-8]. These studies have provided important insights but even striking findings in animal models often fail to translate into useful clinically approaches. Xenograft models have allowed whole human tumor tissue, including TIL, tumor cells and other structural cells to be engrafted into immunodeficient mice and resulted in significant advances in understanding the human tumor microenvironment [9-14]. In one study [14], TIL from implanted tumor were able to migrate to spleen and maintained their characters even after adaptive transfer to another SCID mouse, providing a unique opportunity to investigate the function of TIL and test strategies to eradicate tumor.
The work presented in this study carries animal modeling one step further by simultaneously humanizing the immune system of recipient NOD/SCID/IL-2Rγnull (NSG) animals and challenging them with implanted human tumor cells. This humanized platform provides an opportunity to study the two-way interaction that occurs between human immunity and tumor growth, over time, and to manipulate individual components to test hypotheses and potential clinical impact. As a proof of concept, peripheral blood lymphocytes (PBL) and dendritic cells (DC) were obtained from healthy donors and used to reconstitute NSG animals, followed by implantation with cells from the human prostate cancer cell line, PC3. Resulting tumors demonstrated infiltration by TIL, with a composition and features very similar to those observed in tumor samples from prostate cancer patients, and reciprocal changes were observed in the spleens of tumor bearing animals suggesting both local and distant tumor responses. Serum from these animals contained cytokines produced by human lymphocytes as well as those by tumor, with further evidence of a two-way interaction. Finally, the rate of tumor growth was dependent upon both the presence and composition of the implanted lymphocytes.
Human lymphocytes infiltrate and control tumor growth in huPBL-NSG animals
According to the experimental paradigm, animals were immune reconstituted with a combination of human PBL and DC alone, in combination with the subcutaneous (s.c.) implantation of PC3 cells, or with PC3 cells alone. PBL were prepared from peripheral blood mononuclear cells (PBMC) by depleting monocytes and natural killer cells (expressing CD14 and/or CD16) and activated T cells (CD25+) using monoclonal antibodies (mAbs) as described previously [15]. The resulting PBL consisted primarily of T cells (CD3+/CD56-; 72.7-90.2%), NKT cells (CD3+/CD56+; 2.8-9.8%), B cells (CD3-/CD20+; 3.5-8.0%), and a few NK cells (CD3-/CD56+; 1.0-2.2%). Monocyte-derived DC were CD14-negative and expressed high levels of class I and II major histocompatibility molecules and co-stimulatory molecules [15].
As shown in Figure 1A, the cross-sectional diameters of tumors recovered from huPBL-NSG mice were significantly smaller than tumors from NSG mice that were not immune reconstituted (average 620 ± 233 mm3 vs. 2792 ± 711 mm3; p < 0.05). When the rates of tumor growth over time were examined (Figure 1B), the difference between these two groups did not occur until at least two weeks after implantation, corresponding to the time at which functional immune reconstitution occurs in this model [15]. Immunohistochemistry (IHC) clearly demonstrated the presence of Prostate Stem Cell Antigen (PSCA) positive tumor in both cases and the presence of CD45+ human leukocytes infiltrating tumors harvested from huPBL-NSG animals (Figure 1C). As such, the presence of infiltrating human cells correlated with a reduction in the rate of tumor growth.
Human PBL infiltrate and control tumor growth in huPBL-NSG animals. Six-8-week- old NSG mice were injected with saline alone or with human PBL (1x107) by intraperitoneal injection to produce huPBL-NSG animals. Mice were simultaneously implanted with 2x106 human prostate tumor cells (PC3 cell line) by s.c. injection and tumor growth was monitored over a 4 week period. (A) Final tumor volume (mm3) as measured percutaneously in the animal and corresponding images of tumors dissected from NSG and huPBL-NSG animals at 4 weeks. (B) Tumor growth curves for NSG and huPBL-NSG animals. (C) Recovered tumors were formalin-fixed, paraffin-embedded,sectioned, and stained with antibodies against either human PSCA (top panel, brown stain), to demonstrate tumor cells, or human CD45 (bottom panel, brown stain), to demonstrate infiltrating human leukocytes, and counterstained with Hematoxylin (blue stain). Magnification, x100. Values represent mean of three animals per group ± SD. *p < 0.05. Representative experiment, n = 5 replicate experiments.
TIL recovered from huPBL-NSG animals exhibit a unique phenotype distinct from splenocytes and similar to primary human TIL recovered from patients with prostate cancer
The distribution and features of leukocytes that infiltrate human tumors are distinct from those present in peripheral blood or infiltrating normal tissues. Spleen cells and TIL were therefore recovered at 4 weeks after implantation and examined by flow cytometry. A representative example is presented in Figure 2A and documents the key features of TIL in this model. CD8+ T cells always predominated in TIL and the CD4:CD8 T cell ratio in TIL was always lower than that observed in spleen. While the splenic CD4:CD8 ratio was variable it ranged from 1.8-3.5 fold higher than that in the corresponding TIL (p < 0.005 by paired T test, n = 5 donors). In addition, TIL consistently exhibited a high percentage of cells exhibiting the CD8+/CD56+ NKT cell phenotype (34.02 ± 9.53%) while NKT cells represented only a small minority population within the spleen (5.76 ± 3.24%) and the difference was statistically significant (p < 0.00005, n = 6 experiments using different donors). In addition, the majority of TIL, regardless of whether they were CD4+, CD8+, or CD8+/CD56+ demonstrated the CD69 activation marker, while expression of this marker by splenic T cells was limited. B cells were rarely identified in TIL (<0.5%) nor were CD14+ monocytes (<0.3%), the later findings possibly reflecting the depletion of this population from the PBL used for implantation. While the cell populations and markers were distinctively different between tumor and spleen in tumor-bearing huPBL-NSG animals, the CD4/CD8 ratio and the presence of NKT cells did not differ between the spleens of control huPBL-NSG animals and those bearing PC3 tumors (data not shown). As such, TIL in this model exhibited a unique composition and features. In order to assess whether this recapitulated the features of native TIL we also performed FACS analysis on TIL obtained from patients with primary prostate cancer. As demonstrated in Figure 2B, TIL from prostate cancer also featured a CD8+ predominance with a high percentage of CD8 + CD56+ NKT and expression of the CD69 activation marker. Unfortunately, given the absence of matched spleen samples from patients, the features in human spleen from patients with prostate cancer cannot be directly commented on or compared – one of the important reasons for developing the humanized mouse model.
Phenotype of human lymphocytes recovered from the spleen and tumor of tumor-bearing huPBL-NSG and from primary human prostate cancers. Single cell suspensions were prepared from the spleen and tumor recovered from huPBL-NSG mice at 4 weeks (A) and from a primary human prostate cancer removed at the time of prostatectomy (B). Cells were stained with an antibody cocktail, and then analyzed by flow cytometry for the expression of CD4 vs CD8 (top panel, gated on all cells expressing human CD4 and/or CD8); CD4 vs CD56 (middle panel, gate on all CD3+ cells); and CD4 vs CD69 (bottom panel, gated on all cells expressing human CD4 and/or CD8). The CD8+/CD56+ NKT population is identified by CD4-/CD56+ T cells. Representative experiment, n = 3 experiments.
The human cytokine and chemokine profile in huPBL-NSG animals is altered by the presence of tumor
In contrast to the complexity of human serum, the human proteins present in the serum of huPBL-NSG animals can only originate from the cells that have been implanted and those expanded during the period of engraftment. As such, we hypothesized that serum measurements from these animals would directly reflect their immune reconstitution and its interaction with tumor (Figure 3). Sera were collected at 4 weeks from animals that were implanted with human PBL alone, those receiving both PBL and tumor, and animals that were implanted with tumor alone. hIFN-γ, hIL-10 and hRANTES were all present in huPBL-NSG animals and in huPBL-NSG animals that had been implanted with tumor, but not in animals that received PC3 cells alone. In contrast, while hTNF-α was detected at a low level in one of the huPBL-NSG animals it was detected in all of the huPBL-NSG animals that also had tumor implanted. This difference was not due to the tumor itself, which released no detectable hTNF-α when implanted alone, suggesting an immune cell-tumor interaction. The pro-inflammatory factor IL-8 appeared to be made primarily by the tumor and while IL-6 was released by both immune cells and tumor, its levels were more uniform and robust in reconstituted animals bearing PC3 tumors, again suggestive of an immune cell-tumor interaction. As the sample sizes were small and the distribution of cytokine levels were not always uniform, the mean values of each cytokine for each replicate animal are presented in Figure 3 in lieu of formal statistical analysis. These results suggest that the tumor-bearing huPBL-NSG model provides a sensitive platform for investigating the human cytokine and chemokine interactions relevant to the generation and maintenance of TIL.
Profile of human cytokines and chemokines in the serum of NSG and huPBL-NSG animals in the presence or absence of PC3 tumor implants. Sera were collected from NSG animals 4 weeks after implantation of human PBL and/or PC3 tumor cells and assayed for the presence of human IFN-γ, TNF-α, IL-8, IL-6, IL-10 and RANTES by SearchLight multiplex assay. Representative experiment with each bar representing results from one animal, n = 3 experiments.
Human effector memory T lymphocytes (T-em) are increased in the TIL recovered from tumor-bearing huPBL-NSG animals
A more detailed analysis of T cell subsets was undertaken to evaluate for the presence of naïve (T-naïve, CD45RAhi/CD127hi), central memory T cells (T-cm, CD45RAlo/CD127hi) and T-em (CD45RAlo/CD127lo). As demonstrated in Figure 4, the majority of splenic CD4+ T cells in this model are split between T-cm and T-em, however there is always a recognizable T-naive population. In contrast, the T-em phenotype overwhelmingly predominates in TIL where the T-naive population was essentially absent and cells exhibiting a T-cm phenotype were limited. This overall pattern was similar for both the CD4+ and CD8+ T cells but most striking with respect to the CD8+ subset. While the difference between spleen and TIL subsets was numerically small, especially for the CD4+ population, it was highly reproducible and statistically significant when all mice that had been reconstituted with same PBL donor were compared (p < 0.01 for CD4+ T cells; p < 0.0001 for CD8+ T cells) and this pattern was significant in all experiments, with all donors when normalized by comparing the T-em: T-cm ratio (p <0.05 for both CD4+ T cells and CD8+ T cells). When primary tumors from patients were analyzed, the overwhelming majority of CD3+ T cells were CD45RA-negative (data not shown), which is consistent with the finding from our tumor-bearing huPBL-NSG model. In contrast, the majority of CD3+ T cells recovered from primary human tumors expressed CD127, more consistent with a T-cm rather than T-em phenotype. This likely reflects the sub-acute nature of the tumor-related interaction that occurs in this model, which is temporally different than the interaction between primary human tumors and infiltrating T cells in vivo, which occurs over a much longer period of time.
T- naïve, T-cm and T-em subsets in the spleen and tumor. Single cells were prepared from spleens (A) and tumors (B) recovered from huPBL-NSG mice 3 weeks after implantation and stained with antibodies against human CD3, CD4, CD8, CD45RA and CD127. Cells were gated for expression of CD3 and then analyzed by flow cytometry for the expression of other markers on the CD3+ population. Representative experiment, n = 5 experiments.
Despite the predominance of CD8+ TIL, CD4+ T cells play an essential role in tumor regression in tumor-bearing huPBL-NSG animals
Activated CD8+ T cells and NKT cells with a T-em phenotype predominate in TIL recovered from prostate tumors and in our tumor-bearing huPBL-NSG model. However, animal models have routinely suggested that CD4+ T cell help is essential for effective anti-tumor immunity. As a direct proof of principal, NSG mice were engrafted with either whole PBL or with PBL from the same donor that had been depleted of either CD8+ T cells [huPBL-NSG (−CD8)] or CD4+ T cells [huPBL-NSG (−CD4)]. As shown in Figure 5A, immune reconstitution with CD8-depleted PBL lead to the same control of tumor growth as did the administration of whole PBL. In addition, NSG animals that had been reconstituted with CD4-depleted PBL failed to control tumor growth (Figure 5B). Taken together, these results suggest that CD4+ T cells play an essential role in the immune response to tumor growth even though the majority of cells that accumulate within the tumor exhibit a CD8+ T-em phenotype. To better understand this phenomenon we examined the phenotype of T cells in the spleens and tumors of these animals (Figure 6). As shown in Figure 6A/B, even though CD8+ T cells were extensively depleted prior to implantation (<0.1% CD8+), there was a substantial population of CD8+ T cells (11-15% of total T cells) when cells were recovered from the spleens of reconstituted huPBL-NSG animals. Furthermore, as shown in Figure 6C there was still a preferential accumulation of CD8+ T cells in the tumor where the CD8+ population represented 25-35% of recovered T cells. As others have demonstrated in a variety of models [16,17], the repopulation of single-positive CD8+/CD4- T cells likely represents the rapid and marked homeostatic expansion of small numbers of contaminating CD8+ cells still present in the implanted PBL. On the other hand, the presence of double-positive CD4+/CD8+ T cells may reflect the de novo expression of CD8 that can occur when CD4+ cells are stimulated to expand [18]. As such, while CD4+ cells may be important we cannot rule out a significant contribution of the CD8+ T-em population with respect to controlling tumor growth. A related phenomenon appeared to occur in huPBL-NSG (−CD4) animals (Figure 6 D-F). While CD4+/CD8- cells did not regenerate when CD4+ cells were depleted prior to implantation, there was a striking increase of CD8+/CD4+ double-positive cells in spleen (32.7%) and tumor (27.4%) as compared to animals that received whole PBL where these double-positive cells only represented 8.2% and 9.9%, respectively. Moreover, unlike our consistent observation that the % of single CD8+ T cells was always greater in tumor than in spleen, there was no significant increase of CD8 T cells in tumor (68.1%) vs. spleen (65.5%) in huPBL-NSG (−CD4) mice, suggesting that the failure to expand tumor infiltrating CD8+ T cells contributed to the lapse in tumor control. Collectively, these results suggest a significant interaction between CD4+ and CD8+ T cell subsets is required with an essential role for both in effectively mediating anti-tumor responses.
Role of CD4+ and CD8+ lymphocyte subsets in controlling tumor growth. NSG mice were reconstituted with either 1x107 human PBL (huPBL-NSG) or PBL from the same donor that had been depleted of CD8+ T cells [huPBL-NSG (−CD8); A] or depleted of CD4+ T cells [huPBL-NSG (−CD4); B]. On the same day they received 2x106 PC3 cells implanted by s.c. injection. Tumors were recovered after 3 weeks and individually weighed. Values represent mean of three animals per group ± SD. *p < 0.05. Representative experiment, n = 3 experiments.
Spleen reconstitution and tumor infiltration in NSG animals receiving purified human CD4+ or CD8+ T cells. HuPBL-NSG, huPBL-NSG (−CD8) and huPBL-NSG (−CD4) animals were established as detailed in Figure 5 and implanted with 2x106 PC3 cells by s.c. injection. A & D: The different purified PBL populations, before implantation, were stained with specific antibodies against human CD3, CD4 and CD8 followed by FACS analysis. Results are gated on the human CD3+ cells. Whole spleens B & E and tumors C & F were then recovered after 3 weeks and single cell suspensions stained with specific antibodies against human CD4 and CD8 followed by FACS analysis. Results were gated on all cells expressing CD4 and/or CD8. Representative experiment, n = 3 experiments.
TIL are present in all tumors and represent the endogenous immune response to cancer growth [1-3]. We hypothesized that huPBL-NSG animals might provide a unique opportunity to study the generation and function of human TIL in vivo. NSG mice lack high affinity receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 [19]. This deficit, when combined with their NOD/SCID background, provides an optimal environment for the engraftment of human cells including stem cells, PBL, DC and tumor cells [15,19,20]. Immune reconstitution with PBL is simple and reconstituted animals thrive without evidence of graft rejection or graft versus host disease when studied over a period of one to two months following implantation [15,19]. The combined administration of human PBL and DC, as in the current protocol, results in functional immune reconstitution including the capacity to generate and activate antigen-specific T and B cell responses [15,21]. Our first goal was to determine whether the engrafted human immune system and evolving tumor would interact in a manner that recapitulated the features of human TIL. Indeed, implanting human prostate cancer cells (PC-3 line) into huPBL-NSG animals resulted in a reproducible pattern of T cell trafficking, infiltration and activation that closely matched that observed in human prostate cancers. TIL recovered from tumors were characterized by a skewed CD4:CD8 T cell ratio with predominance of CD8+ T cells and CD8+/CD56+ NKT cells [22,23]. The expression of CD69, a marker of recent activation, was uniformly increased on recovered TIL but not by spleen cells, again recapitulating the pattern associated with human TIL in vivo [23,24]. However, while this model replicated many of the phenotypic features observed with primary human TIL, we observed the expression of a T-em phenotype in our model while TIL recovered from prostate cancer patients had a profile more consistent with T-cm cells. As others have reported a mixture of T-cm and T-em subsets in human TIL, this difference may relate to the sub-acute interaction that occurs in the model over several weeks rather than the chronic interaction of the tumor with infiltrating T cells that can occur over much longer time periods in patients with cancer [23]. Overall, the unique pattern of T cell infiltration and activation observed in TIL as compared to spleen suggests that the growing tumors and reconstituted immune cells are interacting in a manner that recapitulates the essential features of human TIL.
It is well established that TIL can have both prognostic value [25] and act as a target for cancer immunotherapy [26,27]. However, their contribution to the natural history of tumor growth is difficult to directly assess in patients. An important aspect of the humanized mouse model is the capacity to directly compare tumor growth in NSG animals, which lack immune function, with tumor growth in immune reconstituted huPBL-NSG animals generated from the same litter. While the T cell infiltration into the tumor was often subtle on IHC sections, their functional impact on tumor growth was striking. huPBL-NSG recipients were clearly protected from the rapid tumor expansion observed in tumor-bearing NSG controls. However, TIL failed to eradicate the tumor, suggesting a delicate balance between tumor-associated immune activation and tumor-associated immune suppression. TIL contain a variable mixture of activated, functionally tolerant and regulatory T cell and NKT cell subsets and the relative composition of these subsets is likely an important determinant of tumor growth [22,28-32].
In addition to phenotyping TIL and determining their impact on tumor growth, the huPBL-NSG model affords a unique opportunity to manipulate implanted immune cells and/or tumor to address mechanistic questions and test biologic hypotheses. As a proof of principal, we depleted CD8+ T cells from PBL prior to immune reconstitution. Implanted PBL contained only 0.03 to 0.09% CD8+ T cells as measured by flow cytometry (n = 5 donors). Animals reconstituted in this manner were still able to control tumor growth. Conversely, when CD4+ T cells were depleted, tumors grew at an accelerated rate similar to that observed in NSG animals that never received PBL. While these results might seem opposite of expectations, they are entirely consistent with the literature and with the type of immune reconstitution observed within their tumors and spleens. Using mouse models, investigators have described the essential collaboration that is required between CD4+ and CD8+ TIL subsets in order to mediate anti-tumor responses against prostate cancers [33]. While CD8+ T-em cells are the key effector population that mediate tumor killing, they can be skewed by the tumor environment to develop into regulatory and/or tolerant T cells in the absence of CD4+ T cell help. As such, when animals were reconstituted only with CD8+ cells there was limited control of the tumor even though we documented infiltration by CD8+ TIL. Interestingly, when animals were reconstituted with CD4-enriched T cells they still managed to engraft a substantial CD8+ population within the spleen and TIL. It is not clear whether these CD8+ T cells were derived from the hyper-expansion of trace numbers of CD8+ cells or whether other cell subsets were encouraged by the environment to acquire the CD8+ phenotype. However, in the presence of CD4+ helper cells, the level of CD8+ T cell reconstitution that occurs in these animals appeared sufficient to control tumor growth. A number of biological hypotheses regarding T cell subsets, function, and mediators can likely be assessed in this manner. Similarly, key features of the tumor responsible for T cell trafficking, immune activation and immune suppression might be assessed by implanting gene-modified tumor cells or through the administration of neutralizing antibodies directed against specific cytokines, chemokines or other regulatory pathways as suggested in related studies by Ye and coworkers [34].
NSG animals do not make human cytokines or chemokines and the presence of these factors in the serum of tumor-bearing huPBL-NSG animals must have derived from their implanted human tissues. Using serum collected at the end of each experiment we were able to reproducibly measure levels of hIFN-γ, hIL-10, hIL-6 and hRANTES in reconstituted huPBL-NSG animals. As expected, none of these cytokines were detected in NSG animals that were not immune reconstituted (data not shown). Cytokine profiles were entirely different when NSG animals were implanted with tumor alone (PC3-bearing NSG), where only tumor-derived IL-6 and IL-8 were detected in the serum. Of particular interest, hTNF-α appears primarily in the serum of tumor-bearing huPBL-NSG animals with limited detection in tumor-bearing NSG, suggesting that a TIL-tumor interaction is responsible for its production. Similarly, hIL-6 production appeared to increase in tumor-bearing huPBL-NSG animals when compared to levels in huPBL-NSG animals or tumor-bearing NSG. Human studies have reported that increased levels of hIL-6 and hTNF-α are found in prostate cancer patients with advanced tumors, especially in the setting of tumor-associated cachexia [35,36]. While our studies surveyed a range of cytokines in serum as a proof of concept, the tumor-bearing HuPBL-NSG model would also facilitate an analysis of local cytokine production within the tumor or spleen by extracting RNA or homogenizing samples for protein analysis.
In summary, our findings suggest that implanting tumors into huPBL-NSG animals results in T cell trafficking, infiltration and activation within the tumor that occurs in a pattern mimicking that of native human TIL. The presence of TIL is associated with a marked slowing of tumor growth but does not result in tumor eradication, similar to the clinical pattern observed in patients with cancer. Manipulating the T cell subsets administered to these animals directly impacts on the capacity for their TIL to control tumor growth and the effects are consistent with our understanding of the role that different subsets play in tumor immunobiology. Serum monitoring in these animals detects the presence of human cytokines and factors secreted by their reconstituted immune cells, by the tumor, and the changes that occur due to their interaction. This model is simple, adaptable, and allows for highly controlled comparisons. Reconstitution of a mouse with 1x107 PBL requires the equivalent of approximately 10 ml of donor blood and is usually complete within 2 weeks, simplifying the time and expense when compared to studies with purified cord blood stem cells which take months to engraft [19]. More importantly, this model provides access to the tumor, infiltrating cells, blood cells and spleen cells which can be used to investigate both local and systemic effects mediated by the interaction of cancer cells and host immunity. Based on the proof of concept testing reported here, the tumor-bearing huPBL-NSG model appears to have significant potential as a discovery platform for assessing the nature of human TIL and the interactions of TIL with the tumor that ultimately regulate tumor growth. However, even with all of these features this model has potential limitations. In the studies reported here we did not attempt to HLA match the tumor cell line and donor PBL and thus did not attempt to measure the generation of antigen-specific effector cells or identify antigen-specific T cell responses. This limitation can be overcome by HLA-matching of the PBL donor and the tumor, or by obtaining matched PBL and tumor cells directly from a cancer patient. In the latter setting, animals reconstituted with the blood and tumor of a patient would create a completely autologous xenograft model of the anti-tumor immunity that exists within the donor. Another limitation is that even though 10 million PBL are implanted, the reconstituted immune system cannot recapitulate the entire immune repertoire of the donor. However, as has been demonstrated with antigens such as influenza and adenovirus, there is every reason to belief that the model is fairly robust [15,21]. With extended time, a component of graft versus host disease will develop in huPBL-NSG animals [15] and studies should be limited to less than two months following PBL implantation. Alternatively, it has been proposed that NSG animals can be reconstituted with stem cells to produces a more stable, but naïve, immune reconstitution that can last the entire life of the animal [37,38]. However, such an approach still does not address the HLA mismatch with implanted tumor and in the one reported study the results appear very similar to those presented here [39]. A greater understanding of the interaction that occurs between human immunity and human tumors in vivo is needed and this model provides an interesting opportunity to rapidly advance our understanding of human tumor immunobiology.
Six- to 8-week-old NSG mice were bred and housed at UCLA under laminar flow conditions with all food, water (acidified), caging, and bedding autoclaved before use. No antibiotics were administered. All procedures were approved by the UCLA Animal Research Committee.
Culture media and reagents
The human prostate cancer cell line, PC3 (kindly provided by Dr. Reiter, UCLA), was cultured with RPMI 1640 medium (Mediatech, Manassas, VA) supplemented with 10% FBS (Omega Scientific, Tarzana, CA), and penicillin–streptomycin–fungizone (Life Technologies, Carlsbad, CA). Human cells were cultured in RPMI 1640 supplemented with 10% human AB serum (Omega Scientific), penicillin–streptomycin–fungizone, and 10 mM HEPES (Life Technologies). Monoclonal antibody used for human T-and B-cell enrichment included anti-CD14, anti-CD16, and anti-CD25, and for DC enrichment included anti-CD3, anti-CD16, and anti-CD19, all from BD Biosciences (San Jose, CA). Anti-mouse Ig-conjugated immunomagnetic beads were from Life Technologies (M450 Dynabeads; Carlsbad, CA). Fluorochrome-conjugated mAbs against human CD3, CD4, CD8, CD56, CD69, and CD45RA were obtained from BD Biosciences; anti-CD127 from eBiosciences (San Diego, CA); and anti-CD25 from BioLegend (San Diego, CA,).
Preparation of human lymphocytes and DC
Peripheral blood mononuclear cells were separated from the blood of healthy donors by density gradient centrifugation. Purified T-and B-cell subsets were further prepared by negative depletion using a mAb cocktail and anti-mouse Ig-conjugated immunomagnetic beads as previously described [15]. DC were prepared by culturing the adherent fraction of PBMC with 800 IU/ml granulocyte-macrophage colony stimulating factor (GM-CSF; Bayer Healthcare, Seattle, WA) and 63 ng/ml IL-4 (R&D Systems, Minneapolis, MN) for 5 days at 37°C as previously described [40]. Cells were recovered on day 5 and enriched by negative depletion using a mAb cocktail and anti-mouse Ig-conjugated immunomagnetic beads. Purified DC were cultured for an additional 48 hrs with GM-CSF and IL4 and activated during the final 24 hrs with the addition of human MegaCD40L (200 ng/ml, Alexis Biochemicals, Plymouth Meeting, PA) and interferon gamma (IFN-γ; 100 U/ml, PeproTech, Rocky Hills, NJ).
Immune reconstitution and tumor implantation of NSG mice
NSG mice were implanted with 1x107 human PBL (CD3+ or purified CD8+ or CD4+ T cells) in combination with 5×105 autologous DC by intraperitoneal (i.p.) injection. PC3 prostate cancer cells (2x106) were implanted on the same day into the right flank by subcutaneous (s.c.) injection.
Analysis of human leukocyte subsets in the spleen and tumors of reconstituted NSG mice
Single-cell suspensions were prepared from the spleen and tumor by mechanical disaggregation. Cells were stained with fluorochrome-conjugated mAbs and results acquired with a modified FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) with an added 637 nm diode and two associated detectors (Cytek Development Inc, Fremont, CA) and analyzed using FCS Express analysis software (DeNovo Software, Los Angeles, CA).
In vivo release of human cytokines and chemokines
Serum samples were collected by retro orbital bleeding from animals at the time of sacrifice and assayed for the presence of human cytokines (IFN-γ, TNF-α, IL8, IL6, IL10) and a chemokine (RANTES) using a commercial SearchLight multiplex assay (Aushon Biosystems Inc, Billerica, MA).
IHC staining of primary and implanted tumors
Fresh tumor specimens were obtained from patients undergoing prostatectomy through the UCLA Translational Pathology Core Laboratory in conjunction with an IRB-approved consent process. Tumors from NSG mice were recovered at the time of sacrifice, 3–4 weeks after implantation, fixed in 10% formalin and paraffin-embedded . IHC staining with specific antibody to human PSCA and CD45 was also carried out by the Translational Pathology Core Laboratory.
Comparisons between groups were performed using Student's t test, with p < 0.05 accepted as a significant difference between groups. All experiments were performed with replicates and results from at least three separate experiments were pooled for statistical analysis.
NSG:
NOD/SCID/IL-2Rγnull
PBL:
peripheral blood lymphocytes
IHC:
PSCA:
Human Prostate Stem Cell Antigen
T-naïve:
Naïve T cells
T-cm:
Memory T cells
T-em:
Effector memory cells
PBMC:
Peripheral blood mononuclear cells
s.c.:
subcutaneous
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This study was supported by funding from the Department of Defense Prostate Cancer Research Program, Stewart and Lynda Resnick, the Prostate Cancer Foundation, the UCLA Department of Urology, and Core support from the Jonsson Comprehensive Cancer Center. We thank Dr. Reiter for providing PC3 cell line, UCLA CFAR Virology Core for providing human peripheral blood, The Translational Pathology Core Laboratory UCLA for providing human prostate tumor and performing IHC, UCLA Flow Cytometry Core Facility for providing cytometry services, and UCLA CFAR/JCCC Humanized Mouse Core Laboratory for animal housing and maintenance.
Division of Pulmonary & Critical Care, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095-1690, USA
Michael D Roth
& Airi Harui
Search for Michael D Roth in:
Search for Airi Harui in:
Correspondence to Airi Harui.
AH participated in the design of the study, carried out all experiments, performed the statistical analysis, involved in the interpretation of the results and contributed to the writing of the manuscript. MR participated in the design of the study, involved in the interpretation of the results and contributed to the writing of the manuscript. Both authors read and approved the final manuscript.
Human prostate cancer
Tumor infiltrating lymphocyte
Humanized
Basic Tumor Immunology


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10 Best Songs to Learn French Fast
Funny phrases
Untranslateable Words
Reading Hacks
Using Songs To Learn French
Learning French can be a lot of fun if you use fun methods. And singing yourself to fluency is entirely possible. Music has plenty of benefits for language learning. Find out how the best French songs can help you learn the language faster, and a list of great tunes to get you there.
Why French Songs Are Great to Learn
Music is proven to help you learn. It reduces stress and gives you more focus. But, there are even more benefits to learning French with songs.
Music makes learning fun: It's important to have interesting and engaging language learning classes. And music brings a much-needed funk to your lessons.
Easy to incorporate: Listening to music is a flexible activity. You can do it while you cook, work out, or drive. So, listening to French songs to learn can happen anytime in the day.
Finding the rhythm: Every language has a natural rhythm. And using songs to learn French can help you find the rhythm of the French language. The rhymes and beat of the music will help you reach fluency faster.
Enforce spaced repetition: Spaced repetition is the key to language learning. It's the best method to commit your study materials to memory. Repeating and reviewing your lessons is crucial to learn French. But, repeatedly listening to music is a no brainer. If you like the song and get it stuck in your head, your French lessons are constantly repeating.
Build your vocabulary: You can find new words and phrases and build your French vocabulary using the best songs.
Pronounce French better: Sing yourself to fluency! If you sing your favorite French songs out loud, you can quickly learn to get comfortable with the language. There's no better way to practice your pronunciation than by singing.
Discover French culture: Last but not least, these French songs can help you learn about French culture too. Whether the song is about a famous sight or a lost love, you can definitely get a feel for the French mindset.
Best Songs To Learn French
These songs are perfect for French beginners and advanced students alike. This list includes various genres, so you'll definitely find a French song you'll enjoy. They're not only melodic, fun, and beautiful, they're also very popular in France. Knowing these by heart is a great icebreaker when you're talking to a French local. Use these great French songs to learn French fast:
1. Papaoutai by Stromae
This is an extremely popular song in both France and Belgium. The pop song features techno elements, and you may find it in clubs and parties. But don't worry, the lyrics are very easy to understand. And the beat also helps with getting this song stuck in your head. Stromae is also the singer of the international hit song "Alors on danse".
2. Champs Élysées by Joe Dassin
This song has a catchy melody, an upbeat rhythm and uses French words that are easy to understand. Intermediate French students instantly understand and recognize about 70% of the lyrics. Plus, it paints an attractive image of the magnificent, iconic, avenue the Champs Élysées. The enchanting beauty and electric energy of the song and the street immerses the listener in French culture and helps them to experience the beauty of this romance language. Enjoy this classic French song with this lyric video:
3. La vie en rose by Edith Piaf
A classic! You must listen to this incredibly famous French song by one of the greatest French singers of all time. Edith Piaf is a symbol of France. If you're learning French, you should definitely listen to her other famous songs, such as "Non, je ne regrette rien".
4. Quelqu'un qui m'a dit by Carla Bruni
The soft, slow rhythm, simple word and clear pronunciation of this catchy song make it perfect for all levels of learners. The song's quintessential café vibe gives people a feeling of being transported to France. Listening to and singing along with this song makes learning French easier and more fun.
5. Sympathique by Pink Martini
This song seems like it came straight out of a French textbook. The song is very easy to understand, and the music video purposefully features French vocabulary with translation. But don't be fooled, this is a genuine song. Make the best of your French lessons by listening to it and watching the music video.
6. Il est temps by Kyo
If you ever wondered what a good French boyband sounded like, Kyo is an excellent choice. "Il est temps" is a pop song about love and relationships. The clear voices can certainly make the song easy to understand, even for beginners.
7. Dernière Danse by Indila
This song is extremely famous and popular in France. You may come across it at parties or clubs. It's slow enough to easily understand, and the melody is beautiful too. Watch out, this song may get stuck in your head! The beat and passion of the lyrics are infectious.
8. La Vie Ne M'Apprend Rien by Daniel Balavoine
Daniel Balavoine is a French rock legend. Unfortunately, he died well before his time at 33 in a helicopter accident. His soothing voice lives on in this beautiful song.
9. Aussi Libre Que Moi by Calogero
Pop-rock fans can rejoice: Calogero is an excellent French artist in the genre. This song features a nice beat, fun rhythm, and easy lyrics. You may find yourself dancing to this catchy French tune.
10. La Tribu de Dana by Manau
If you're up for the challenge, try to learn La Tribu de Dana by heart. This French old-school hip-hop song features a lot of rap. Don't worry, with plenty of listens, you can definitely remember the whole lyric, even the fast parts. The catchy tune also helps with that.
How to Learn French Best with Songs
Before you dive into these great French songs, make sure you're doing it right for language learning. Follow these simple steps to maximize your French language learning success:
Read the lyrics: Read the lyrics as you listen to these French songs. This will enhance your reading and listening skills faster.
Look up unfamiliar words: Search for the meaning of new vocabulary. But, if you can, try to guess the meaning of unfamiliar words from context.
Sing French songs: Don't be shy. The best way to reach fluency fast is to speak. And singing is almost the same thing. Don't worry about being on-key either. You're learning French, not competing in X-Factor.
Make it a habit: The more you listen and practice, the better. Spaced repetition and practicing your pronunciation is the best way to reach French fluency fast!
Learn French with More Than Just Songs
As much fun as singing your way to fluency is, you can't learn French with just songs. You need a reliable language learning method. One that gets you speaking from the first lesson. One that gives you useful vocabulary, and never makes you feel like you're learning. And that's exactly what OptiLingo is.
OptiLingo is a language learning app like no other. It actually brings you results. This app gives you high-frequency phrases, so you'll learn exactly how the people speak. And, you'll never even realize you're learning. With built-in spaced repetition, you're guaranteed to remember your lessons. Try OptiLingo today to discover how effective and fast learning French can be!
How Long Does it Take to Learn French?
French Family Culture and Vocabulary
Everything About the French Language
Public Transportation in France

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Arabica capped a surprising session up 360 points at 110.25. The market was under pressure early, falling into negative territory under the weight of broad based Ag selling. Sugar saw a 4500 lot sell at 6:30, Corn saw 7k lots around 9:30, and KC was hit with 1000 lots at 7:45. A stronger dollar and weaker BRL should have been problematic by all typical standards. The COT revealed the funds at their least short since September 2017. Nevertheless, while the all these factors persisted, coffee turned upward around 10am and never looked back. Intraday lows came in right around the 26dma. The source of the rally remains a mystery, but at an estimated 20k lots of outright volume, it was real buying. Option volumes were strong, led by 3500 U 100P on the nose, or 68% of the total OI. Around ½ of those came via the U 105 / 100 1x2. Robusta closed $10 higher at 1434, waiting until minutes before settlement to rally into positive territory.
Morgan Stanley Coffee Report - July 8'19
While temperatures were colder than advertised, cracking the freezing level in areas of the coffee belt, most traders (and meteorologists) dismissed the likelihood of meaningful crop damage, setting the stage for a 470 point loss (106.40 KCU settlement). Initial returns would not have suggested the day in store; prices gapped up from Friday's close on the opening, posting a 114.65 high (+415 from last week's final print) in the first two minutes. Two waves of prominent selling would arrive between 7:30 and 7:40am EDT however, fully altering the complexion of the market. The second, larger, sell order came in just shy of 1800 lots in KCU, sending prices plunging over 400 points and instigating a 30 second Ice trading halt that went unnoticed by many. Prices snapped back after trading resumed and futures were quiet and orderly for most of the balance of the day, with occasional bouts of selling interrupting the calm. Interestingly, outright volume for the day was fairly small by any standard, but particularly given the magnitude of the move, coming in at an estimated 16,412 lots in Arabica, backing up our perception of thin liquidity. London fell $18 to 1426, and interestingly saw a similar sell order to KC at the same 7:40am EDT time. Far less volume was needed to send prices spiraling lower, 280 lots, though the degree of travel was also less – below 1c equivalent.


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Once you accomplish that, it is easy to order HGH through legitimate channels. What is a legitimate channel? You only want to get HGH injections from a licensed US pharmacy. The companies that advertise HGH for sale online are often warehouse type operations that engage only in sales and not medical treatments. Their websites offer little in the way of information about health-related conditions and how HGH can help.
Before you purchase HGH injections in this manner, do your research. The wrong choice could have dire consequences for your health.
Is It Safe to Purchase HGH Online?
Because there are so many "black market" websites engaged in the sale of online medications, it will be difficult not to click on at least a few of them in your search. Why is it not safe to purchase HGH online from these companies?
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With a plethora of affordable treatment options, knowing where to purchase HGH injections with confidence by contacting Greenberg Health is always the right choice for human growth hormone therapy.


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Baptist Press Archive
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High court orders review of 'Janet Jackson case'
by Staff, posted Tuesday, May 05, 2009 (10 years ago)
WASHINGTON (BP)--The U.S. Supreme Court May 4 ordered the U.S. Third Circuit Court of Appeals in Philadelphia to reconsider its ruling in the 2004 Super Bowl halftime case involving Janet Jackson in light of the high court's recent decision on fleeting obscenities.
Last year the Third Circuit overturned the Federal Communications Commission's $550,000 fine against CBS, saying the FCC deviated from its 30-year practice of fining indecent broadcast programming only when it was so "pervasive as to amount to 'shock treatment' for the audience."
But on April 27, the Supreme Court narrowly upheld the ability of the FCC to prohibit certain indecent language on broadcast television, even if it is a one-time instance. In that 5-4 majority opinion, the court reversed a lower court ruling by the Second Circuit Court of Appeals that had gone against the FCC.
The Supreme Court's single-sentence order Monday remanded the Super Bowl case back to the Third Circuit "for further consideration in light of FCC v. Fox Television Stations, Inc.," the name of the most recent Supreme Court decision.
Tim Winter, president of the Parents Television Council, applauded the Supreme Court's most recent order.
"Today the Supreme Court is siding not only with families, but with Congress and the overwhelming will of the American people in asking the Third Circuit to take another look at its absurd ruling," he said in a statement.
"Just last week, the high court affirmed the FCC's ability to sanction a broadcaster for so-called 'fleeting' violations of the broadcast decency law. Today's decision is absolutely consistent with last week's announcement," Winter said.
The Third Circuit last July said the Super Bowl incident was a "deceitful and manipulative act that lasted nine-sixteenths of one second" and that CBS was not responsible "for the acts of Janet Jackson and Justin Timberlake, independent contractors hired for the limited purposes of the halftime show." Jackson and Timberlake sang during the halftime show, with Jackson infamously exposing herself at the end of the performance.
Chief Judge Anthony Scirica, who wrote the circuit court opinion, said the FCC failed to demonstrate that CBS willfully helped orchestrate the indecency, and such willfulness is "the constitutional minimum showing for penalizing the speech or expression of broadcasters." The $550,000 fine represented the maximum $27,500 levied against each of the network's 20 owned-and-operated stations.
But in the April 27 Fox decision, the Supreme Court held the broadcaster to a higher standard, noting that children were watching the awards programs in question.
"Here it suffices to know that children mimic the behavior they observe -- or at least the behavior that is presented to them as normal and appropriate," Supreme Court Justice Antonin Scalia wrote for the majority. "Programming replete with one-word indecent expletives will tend to produce children who use one-word indecent expletives. Congress has made the determination that indecent material is harmful to children, and has left enforcement of the ban to the Commission." Read More
GCR: Signers of document say 'Great Commission Resurgence' needed
by Mark Kelly, posted Tuesday, May 05, 2009 (10 years ago)
NASHVILLE, Tenn. (BP)--The "Great Commission Resurgence" declaration being circulated by Southern Baptist Convention President Johnny Hunt represents a heartfelt effort to deal with the challenges churches face with witness and ministry in today's culture, four men who signed the declaration have told Baptist Press. Read More
GCR: Tone, focus, clarity of declaration questioned by non-signers
NASHVILLE, Tenn. (BP)--Reaction to the "Great Commission Resurgence" declaration in various quarters of Southern Baptist life includes questions about its tone, focus and lack of clarity. Read More
GCR: Merge mission boards, chairman of North American Mission Board says
JACKSONVILLE, Fla. (BP)--The trustee chairman of the North American Mission Board believes the "Great Commission Resurgence" declaration creates an opportunity for dialogue about an issue he believes needs to be addressed: a merger of the SBC's two mission boards. Read More
GCR: GPS rally cry will unite Southern Baptists around Great Commission
by Geoff Hammond, posted Tuesday, May 05, 2009 (10 years ago)
ALPHARETTA, Ga. (BP)--When the North American Mission Board began discussing a new denomination-wide evangelism initiative with our state and local partners back in 2006, we had no idea what the world would be like in the days leading up to the launch of the emphasis. Back in 2006, jobs were plentiful, credit was flowing, mortgages were easy to get and the bull market seemed like it would never end. Read More
GCR: My bloated bureaucracy
by Jimmy Barrentine, posted Tuesday, May 05, 2009 (10 years ago)
DES MOINES, Iowa (BP)--I begin with a serious word. The remainder of the document will take on a less sober tone. Read More
GCR: 'Great Commission Resurgence' needs Great Commission partners
by David S. Dockery & Timothy George, posted Tuesday, May 05, 2009 (10 years ago)
JACKSON, Tenn. (BP)--Over the past two years we have called for consensus, renewal and bridge building in our shared work across Southern Baptist life. We applaud the fact that the spirit of that call is reflected in so many aspects of the recently released statement, "Toward a Great Commission Resurgence," by SBC President Johnny Hunt. Read More
GCR: Akin discusses its history, intent
by James A. Smith Sr./Florida Baptist Witness, posted Tuesday, May 05, 2009 (10 years ago)
WAKE FOREST, N.C. (BP)--Although the declaration, "Toward a Great Commission Resurgence," is now the "property" of Southern Baptist Convention President Johnny Hunt, the statement with more than 1,500 endorsers is the handiwork of Danny Akin, president of Southeastern Baptist Theological Seminary. Read More
TRUSTEES: GGBTS budget reduced by 4.7%
by Phyllis Evans, posted Tuesday, May 05, 2009 (10 years ago)
MILL VALLEY, Calif. (BP)--Trustees of Golden Gate Baptist Theological Seminary adopted a reduced 2009-10 budget during their April 27-28 meeting at the seminary's Northern California Campus in Mill Valley.
Trustees also approved a new master of missiology degree program and received reports on the recent visit regarding the seminary's ongoing accreditation by the Western Association of Schools and Colleges (WASC) and the Association of Theological Schools (ATS).
The $10,028,000 budget ... Read More
FIRST-PERSON: What is your perspective on life?
by John L. Yeats, posted Tuesday, May 05, 2009 (10 years ago)
ALEXANDRIA, La. (BP)--For every individual on planet earth there is a perspective about what life is all about. Read More


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Wall Street surges in best rally since March 2009; oil soars
Sharemarket
By Vildana Hajric & Jeremy Herron
December 27, 2018 — 8.46am
Wall Street staged one of the biggest rallies of the 9½-year bull market after coming within points of seeing it end, with major indexes surging at least 4.9 per cent. Crude jumped almost 10 per cent.
All but one member of the S&P 500 advanced, the Dow Jones Industrial Average jumped more than 1050 points and the Nasdaq 100 rallied 6 per cent. Each gain was the best since 2009, the year the longest bull market on record began.
Dead cat bounce or phoenix rising from the ashes? US markets staged a massive rally overnight. Credit:Bloomberg
Consumer shares paced the rally; Amazon jumped 9.5 per cent after reporting record Christmas sales. Each member of the FAANG cohort rallied at least 6.4 per cent, while energy producers surged as crude powered past $US46 a barrel.
US President Donald Trump said a day earlier that the rout that took stocks down 19.8 per cent from a record provided a "tremendous opportunity to buy".
Investors also welcomed Kevin Hassett's assurance that Jerome Powell's job is "100 per cent" safe. Oil's best rally since 2016 added to the equity surge.
"It was probably a pretty good retail-oriented holiday and that probably has a lot to do with what's happening today," said Kim Forrest, a senior portfolio manager at Fort Pitt Capital Group.
"The thing that the Fed chairman won't be axed, that has a lot to do with everyone being happy Powell gets to keep his job and that the turmoil about this has abated for today. You have the market leaning one way or the other, and it can often do what it's doing today, which is go higher. On Monday the market leaned lower. It's an outsize move."
Stocks are looking to stop one of the most miserable Decembers on record, as a host of headwinds combined to drag down America's benchmark index.
A reminder that consumers - a key part of the American economy - remain on solid footing helped soothe anxiety created by fears of a global slowdown and personnel churn in the US administration. Volume was in-line with the 30 day average at this time of day.
Hassett was the latest government official to try to calm the markets after Bloomberg's report on Friday that President Donald Trump asked about firing Powell. Steven Mnuchin was criticised for saying he called bank chiefs to gauge liquidity. Trump expressed confidence in Mnuchin on Wednesday.
Crude surged, the greenback was stronger versus its major peers and Treasuries fell. Exchanges throughout Europe remained closed for the holiday.
Elsewhere on Wednesday, Japanese equities closed higher on a wave of late buying after fluctuating throughout the day. Korean shares tumbled after a holiday, and Shanghai stocks fell for a second day. Markets in Australia and Hong Kong were closed.
West Texas Intermediate crude rebounded to trade above $US44 a barrel. The offshore yuan was little changed after China released new rules promising to treat all companies equally, the latest positive step on the trade and investment front since further US and Chinese tariff hikes were paused.
"There's a lot of uncertainty in the short term and that makes sense," Gershon Distenfeld, AllianceBernstein co-head of fixed income, said on Bloomberg TV.
"We're going to have a lot of volatility. But this base case of 'the world is coming to an end', just given the fundamental data out there, doesn't make any sense."
Here are the main moves in markets:
The S&P 500 Index rose 4.96 per cent as of 4pm in New York, after falling within two points of a bear market earlier in the session.
The Nasdaq 100 surged 6.2 per cent and the Dow Jones Industrial Average rallied 1086 points.
The MSCI Asia Pacific Index gained 0.2 per cent, the first advance in a week and the biggest rise in more than a week.
The MSCI Emerging Market Index fell 0.2 per cent.
The Bloomberg Dollar Spot Index climbed 0.3 per cent.
The euro fell 0.3 per cent to $US1.1353.
The British pound fell 0.3 per cent to $US1.2637.
The Aussie dollar rose 0.45 per cent to US70.68¢.
The Japanese yen weakened 0.9 percent to 111.284 per dollar, the first retreat in almost two weeks.
The yield on 10-year Treasuries gained five basis points to 2.79 per cent.
The two-year rate added five basis points to 2.60 per cent.
West Texas Intermediate crude was up 9.5 per cent at $US46.59 a barrel.
Gold fell 0.2 per cent to $US1268.80 an ounce.
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Ceva Animal Health wants to give our clients, suppliers and media partners immediate access to any timely and relevant news.
On Thursday 27 October 2016 Stuart Tinney and I gave a talk at Hawkesbury Riding Club in Clarendon.
Ceva was hugely excited that our long term sponsored rider Stuart Tinney was a Bronze medallist at the 2016 Rio Olympics.
Every year for over 15 years Ceva (formerly Nature Vet) has hosted the Tuesday night Causal dinner at the Equine Veterinarians Australia (EVA) Bain Fallon Conference.


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By Maxwell Flynn 5 days ago
Welcome to Hard Time Blues: The World of Pro Wrestling from Merry-Go-Round Magazine! It's a great time to get into professional wrestling. In each Hard Time Blues feature, MGRM contributors Luke Phillips and Maxwell Flynn will be exploring the wild, weird, and wooly world of the squared circle, and all of the angles, feuds, gossip, and evil wizards therein.
This week, Maxwell Flynn highlights West Coast Pro's first show of 2023, CAN YOU WORK FRIDAYS? (available for replay on IndependentWrestling.tv), and asked wrestlers the question: "Why do you love professional wrestling?"
I like professional wrestling for the drama, the showmanship, and the spectacle. Every match tells a different story, usually a small piece of a larger story—that's told over weeks, months, or even years. Some people watch for the looks, the costumes, makeup, and presentation of characters; others watch purely for the athleticism.
On January 6th, I had a chance to speak with some wrestlers before West Coast Pro's first show of the year, CAN YOU WORK FRIDAYS? about what they love about professional wrestling, and drill into the connects all of us—fans and wrestlers alike—to the sport.
Alpha Zo, a member of local favorites The Conglomerate, loves professional wrestling because it's "a stress reliever." "While the fans are enjoying what they're watching, we're enjoying it ourselves," he said. He appreciates the feedback he receives from the crowd reaction: "If we're doing something right it's like 'Yeah, I fuck with that!' but if we do something wrong we can notice it and for the next show [we're] not gonna do that. You make sure the crowd is pleased and can go home happy." After a losing effort in an absolute brawl of a match against the West Coast Wrecking Crew and "Filthy" Tom Lawlor, The Conglomerate were attacked by three new faces in WCP. Representing Southern California, Adrian Quest and Los Suavecitos made their presence known before being chased off by Zo, bringing order back to the show and standing tall before the crowd that loves him.
West Coast Pro announced their newly formed Women's Championship last year and are getting closer to crowning the inaugural champion. At CYWF, Sandra Moone successfully faced off against Vert Vixen for a spot in that deciding match, which will take place at WCP's March 4th show."It's a unique way for self expression," Moone says reflecting on the sport. "I feel like being able to put myself on a larger stage, like professional wrestling, allows the fans to put themselves to you, where they don't have to worry about anything else. They're just mesmerized by these stories. It's really about the art of it."
🎮 P L A Y E R O N E 🎮
Lfg again @WCProOfficial pic.twitter.com/3KYL89FDSG
— VertVixen – Player One 🎮 (@VertVixen) January 19, 2023
I've seen "The Bounty Hunter" Bryan Keith wrestle a handful of times over the past year and I'm always left in awe as he puts his all into each performance, taking his opponent to their limit. At CYWF he kicked off a monumental weekend facing off against Black Taurus, a mountain of a man, in a spectacular match pitting cowboy against bull. He went on that weekend to participate in Pro Wrestling Guerilla's 17th Battle of Los Angeles, a prestigious tournament in the independent wrestling scene. With that in mind, fittingly the word he used to describe what he loves about the sport was "Unity." "The fact that I meet people that on a regular day basis I wouldn't interact with at all, and wrestling is what brings us all under one roof." Keith is someone to keep your eye on, with an insane amount of star potential it's only a matter of time before he's doing what he loves on a worldwide stage.
That idea of unity and community—professional wrestling bringing people together—was a common thread with the people Lazarus, who (as his name would imply) is some sort of ghoul, zombie, or perhaps even a man struggling with the effects of being reincarnated, has "always loved the characters […] all of the different kinds of people that come through, [the] masks, […] there's just so many different kinds of people; and when it comes to traveling you get to meet all those people and all the people that you work with are all characters in themselves and everyone is unique."
At CYWF Lazarus went head to head with Robert Martyr, a violent young man who is out to prove himself in the ring. "I had a childhood of negligence and that lack of attention stuck with me for a long time. When I make people feel and react and care I get a high off that." Martyr said. "When you're a kid and nobody pays attention to you you want that attention that you wouldn't otherwise see. When I did boxing nobody gave a fuck, I was getting my ass beat and nobody could give two shits about it but I do pro wrestling and people seem invested." Lazarus vs. Robert Martyr was a back and forth struggle with both men pulling whatever they could out of their repertoire in an attempt to take the victory. While Martyr came out on top it was an impressive showing for both and I'm excited to see where each of their career's take them.
Nick Wayne, son of wrestling legend Buddy Wayne and seventeen year old wrestling prodigy, was presented an AEW contract back in February of 2021. After making an appearance on AEW DYNAMITE on January 4th, he competed in a match against modern wrestling icon Alex Shelley at CYWF. "Something that's very special about [professional wrestling] is the emotional attachment you can make with fans, or just regular people that aren't wrestling fans. There's always that group of people that come to shows that are coming for a good time, they don't know [anything] about wrestling, and when you can create an attachment with them to where they want to see you win and they are interested in you and your work I think it's something very fascinating that we can do. Like pull those strings with people's emotions like that and get them to really believe this stuff. I just think it's something that's super special that professional wrestlers can do that not a lot of people in other entertainments can do." Picking up the win over Shelley, it's clear that Wayne's pedigree, mind for professional wrestling, and connection with the fans has him on a fast track for stardom.
Alex Shelley vs. Nick Wayne
West Coast Pro Can You Work Fridays?(1/6/23)
Nick Wayne at 17 did a better job selling his knee in this match than Seth Rollins ever has in his entire career
Also Alex Shelley is a God pic.twitter.com/qKa2yOBPba
— Pete (@pinklightsticc) January 14, 2023
The main event at CYWF saw newly crowned West Coast Pro Heavyweight Champion Titus Alexander defending his title against rising talent Alec Price in a thrilling match that drew the love, and hate, from every person in the crowd. Representing Massachusetts and playing off of the West Coast vs East Coast rivalry, Price was undoubtedly the villain in this matchup. Price loves professional wrestling "because it mirrors life, like the ups and downs. […] to become a really good professional wrestler you have to grow not only as a human but as a pro wrestler and that comes hand in hand." He recalled that when he first got into wrestling he "was a cocky piece of a shit," that "from where [he] came from [he] had to be like that." After getting checked by an OG from the Massachusetts scene, Mike McCarthy, he was (in his own words) "humbled." "I went from not knowing what I was gonna do in my life to realizing my dream and finally realizing, finding what I was put on this earth to do."
Apparently he was put on earth to talk shit to the crowd and push Titus Alexander to the edge. On the other side of the spectrum, Alexander puts off a cold demeanor in the ring—a cocky attitude with a smirk of confidence. But underneath the surface he has a true passion for what he does: "Being in the ring, being center stage in front of all the fans, living your dream, I feel like it's unreal. It's like you almost switch out of yourself; it's a chance to be someone else, someone you're not maybe." Both of these young men have an air about them that says they know exactly how good they are, and believe me they really are that damn good, and to see them face off so early in their careers, with so much ahead of them, is very exciting from the perspective of a fan.
The world of professional wrestling has something for everyone, even those who are doing the wrestling. It contains multitudes; whether you like submission clinics, hard hitting slobberknockers, high flying spectacles, or technical masterpieces; if you like character driven storylines or just a good old underdog rising to the top. Over recent years it has seen a resurgence, and with that has come a closer camaraderie amongst the fans and wrestlers. Professional wrestling is like a family—a weird family with bull-men, ghouls, violent lunatics, and even "real life dinosaurs," but like Bryan Keith said: there is unity there.
CAN YOU WORK FRIDAYS?
West Coast Pro
Maxwell Flynn
Maxwell Flynn is an avid professional wrestling fan. He grew up watching WCW and bootleg ECW tapes; his current favorite promotions are AEW, AAA, and West Coast Pro. When he's not watching wrestling he's grilling or playing fetch with his dog.
Hard Time Blues' Favorite Matches of 2022
West Coast Pro Show No Signs Of Slowing Down During Ride The Lightning
Hard Time Blues vs. Jobber
West Coast Pro's Inaugural West Coast Cup Captured All The Brilliance Of Wrestling
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View all Hard Time Blues: The World of Pro Wrestling


New Chelsea manager Antonio Conte has stated that he won't rush Kurt Zouma back into the team.
He said that the player needs time to recover following his injury and to get back his fitness.
The French player sustained the injury in a match against Manchester United and had been recovering since then.
The prognosis was that Kurt Zouma would have been out for six months, but now it is believed that the player will not be back until the end of the year. Antonio Conte believes that after such a long time from playing, he will need some time to adapt and to get back into the game.
Zouma is currently with Chelsea in Austria as the London club prepares their season. Antonio Conte said that he knows that the player is desperate to play with the first team, but he stated that he needs to be patient. He stated that he will be monitoring his progress closely and that he will allow the player to make his return gradually. He said that the Frenchman is a valuable member of the team and that he will not risk losing him to another injury.
Antonio Conte is confident that his Chelsea side will be able to compete for the title next season. He believes that he has a strong squad and that the players have a chance to challenge for the title. He has told his players that they should be prepared to give 100% in every game if they want to stay in the team. He said that there is no shortcut for hard work and that if the team wants to improve they will need to train hard. It is believed that Chelsea will be looking to add in a few more players before the start of the season so as to bolster the squad.


Insurance companies know the chaos following an accident is added stress on top of an injury. Before you accept a settlement check, make sure it's one that covers all the costs of your recovery. Cueria Law Firm handles accidents on land, water and in the air. Contact us today at (504) 525-5211 for a free consultation.
Insurance adjusters may act as if they are looking out for you, but the reality is that it is their job to offer you the lowest possible amount for your injuries.
We defend injured drivers against trucking companies.
What Uber and Lyft users should know about accidents.
Helicopter accidents have extreme costs for recovery.
Each of the above situations can result in a serious or even fatal injury for the unsuspecting driver or passengers involved. The job of a personal injury law firm in this case is to help the injured party establish that another party was liable through negligence. This allows the injured parties to make a claim against any insurance company or other liable parties in order to get the money they need to recover from their injuries.
Insurance companies fight these battles each day, and many times they'll get away with short changing the injured party who doesn't realize they can make a claim for more than just initial medical bills. Seeking legal guidance from a personal injury attorney like the transportation accident attorney at Cueria Law Firm, LLC, can mean the difference between fully recovering from your injuries and suffering, or stressing each day about the pain you are in and the money you owe for medical treatment.
Whether you're a driver, motorcycle rider, cyclist, pedestrian or passenger in a car, train, bus or plane, it's important to know what to do in the event you are injured in an accident. The moments following an accident can see chaotic, especially if you have a major injury. But these moments are also what could make or break your personal injury case.
Contacting emergency responders will mean two things: first, you'll get an ambulance and paramedics who can assess your injuries. Secondly, you'll have a police officer respond who will write a report documenting the facts of the accident and the contact information of all involved parties. This police report may also include the officer's opinion of who was at fault, which can be helpful if your insurance claim is denied.
In some cases, such as accidents which occur on private property or on the water, it may not be possible for you to get a police officer to respond. Ask if there is an incident report that can be filed by the owner of the property (this is usually the case for businesses and organizations). If that is not an option, do your best to collect the information from the involved parties including the other driver, any passengers, and any witnesses of the accident. If you have a camera capable cell phone, take photos of the damage and injuries caused by the accident.
In the moments after a traumatic event such as a car accident you may not be able to properly assess your injuries. If you go about your day after sustaining a minor injury you could cause further damage. It's best to go to an emergency room even if you feel okay.
If you file a claim or if the other party involved in the accident does, you will be contacted by an insurance company. They will have questions regarding your accident, including your injuries and personal information.
When you are offered a check to cover you medical treatment, it is in exchange for your right to make any future claims against their insured. While it may be tempting to accept a three or four figure check in the days following your accident, you may be entitled to much more. An experienced personal injury attorney will be able to review the facts of your case and advise you on which damages you are able to claim based on the cases they have negotiated in the past.
In the most severe accidents, injuries may result in the death of a loved one. In these cases, the loved ones of the deceased may have the right to make a wrongful death claim. These claims are reserved for the closest kin (usually a child or spouse) but can be made by the parents, siblings, or any dependents of the person who died in the accident. These claims will recover damages for loss of society and companionship, as well as to cover medical costs or funeral costs incurred by the deceased. To find out if you have a wrongful death case, you should contact an experienced personal injury attorney.
If you or a loved one have been injured in a transportation accident, contact Cueria Law Firm, LLC, for a free consultation today. Our experienced team has dealt with every type of transportation accident including auto accidents, truck accidents, bus accidents, rideshare (Uber & Lyft) accidents, helicopter and plane accidents, railroad accidents and pedestrian or cyclist accidents. We have a solid understanding of the insurance company tactics used to justify a lower claim than the victims of a personal injury accident deserve. Cueria Law Firm, LLC, will get you the compensation and justice you deserve to move on with your life.
You can contact Cueria Law Firm today to request a free consultation on your accident by calling (504) 525-5211.
Tell us about your accident and injury. We can help.


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Telecom and Telefonica question Telmex decision
According to local newspaper Los Andes, Telecom Argentina and Telefonica de Argentina have questioned the government's decision to award Mexican telco Telmex a contract to build a communications network for the police. The two companies claim that Telmex is not the best operator for the job, but the Mexican telco counters by saying that it has a larger presence in the country than them, through its subsidiary CTI. Telmex is ready to sign the USD18 million contract with the government and the new system is expected to be operational by the end of this year.
Argentina, Telecom Argentina (incl. Cablevision), Telefonica Argentina (Movistar), Telefonos de Mexico (Telmex), Wireline


My magic show is non-stop, age appropriate comedy magic. My balloon show amazes people at what can be built from balloons. I typically do birthday parties, school shows, Blue/Gold banquets, YMCA/YWCA camps and the like. Whether its a birthday party, a banquet, or a school, you will hear the children laughing and see them grinning continuously. One highlight of the show is my rabbit puppet who is convinced HE is really in charge of the show. Every child squeals with delight at the appearance of my REAL RABBIT, Wilbur, who knows HE is the real star. All the children love to participate as they help the elusive Hiram hide from the poor magician. For birthday parties, I customize the show to the age of the participants. The show for the 3-5 year old audience is different from that for the 6-9 year old crowd. In addition, I have a workshop aimed at older children. The workshop teaches both how to do a series of tricks AND how to present them. Frequently people at schools and churches wonder if I have more than shows that compliment what they are teaching. I am happy to say that I do. I currently have a set of shows whose topics include, but are not limited to my: Anti-Bullying show Safety show, Dental Hygiene show, Lean Green Eating Machine (Healthy eating) show, Self Esteem show, I love America show, as well as Holiday theme shows for Halloween, Christmas, etc.
People at schools and churches frequently ask Bjorn the Magician if he has more magic shows that compliment what they are teaching. This educational kids entertainer is happy to say that he does.
This kids magician has a set of shows whose topics include, but are not limited to his: Safety, Dental Hygiene, Lean Green Eating Machine (Healthy eating), Self Esteem, I love America, as well as Holiday theme magic shows for Halloween, Christmas, etc.
If you are an educator and want a wonderful and educational kids magic show that supports your curriculum, then call this kids magician today.
Why do you need a kids party entertainer when hiring a childrens party magician for your childrens party?
When you have a large number of children at a birthday party expecting entertainment, they can become very excited. If that entertainment does not happen quickly enough, then the children may grow bored, or begin to entertain themselves. That might not be entertaining for the other kids or their parents.
You need a kids party entertainer who is able to connect with the children and capture their attention with zany magic tricks, hilarious stories, and incredible puppetry. When I produce a real live magician's bunny, the children laugh and applaud, and focus on him for a long time.
Our kids party magic shows delight children and give the parents some time to relax a little because we are entertaining both the children and the parents. It also gives you the opportunity to take some great photos your guests enjoying your kids birthday party show.
Bjorn the Magician is a zany and witty Childrens Entertainer who will amaze the kids, their parents, and you with his delightful magic tricks, wacky stories, and colorful balloon creations. Bjorn knows the kids are the real stars of his children's magic shows.
Because Bjorn is able to connect and communicate with the children, kids eagerly volunteer to help him with the magic show. He responds well with each child's personality and is able to give a unique show for every audience.
Childrens Entertainer, Bjorn the Magician, has been professionally entertaining family audiences for over 25 years at hundreds of events.
Bjorn has entertained at many different types of functions, from local kids birthday parties to government functions.
He will work with you to make sure your kids party event is truly one of a kind.


text:
Q: parsing both Python and click command line options Often I want to profile a CLI program built with Click, but I can't figure out how to parse both the Python command line options and the click command line options/arguments.  For example, if my script took a single option and two arguments, I can run it fine like:
python add.py --verbose 1 2

Similarly, when I profile a simple (non-Click) script, I can do this:
python -m cProfile -o stats.txt add_no_click.py 1 2

but when I want to profile the Click script, I get this:
python -m cProfile -o stats.txt add.py --verbose 1 2
Error: no such option: -o

I know I must be missing some documentation out there.  For what it's worth, I'm using Python 2.7 on Windows 10 64-bit, although I doubt it has a bearing on the answer.

A: Sorry for the self-answer, but hope this might help others.  The stupid thing I did (and that I didn't show in the question above) was forgetting to call main with just sys.argv[1:].
Sample program:
import click

@click.command()
@click.option('-v', '--verbose', is_flag=True)
@click.argument('a', type=click.INT, required=True)
@click.argument('b', type=click.INT, required=True)
def main(verbose, a, b):
    if verbose:
        print('The answer is {}'.format(a + b))
    else:
        print(a + b)

if __name__ == '__main__':
    import sys
    main(sys.argv[1:])

Running it:
> python -m cProfile -o foo.stats add.py --verbose 1 2
The answer is 3

and the profile information is written to foo.stats.

redpajama_set_name: RedPajamaStackExchange


I was searching for a Property and found this listing (MLS® #1615447). Please send me more information regarding 3551 Hudson Hills Ln Unit 24, Deerfield Twp., OH, 45040. Thank you!
I'd like to request a showing of 3551 Hudson Hills Ln Unit 24, Deerfield Twp., OH, 45040 (MLS® #1615447). Thank you!
MLS® 1615447 is a Single Family home located at 3551 Hudson Hills Ln Unit 24 in Deerfield Twp. OH. The property features 5 bedrooms and 6.00 bathrooms. This property is currently listed for $659,000.
This property comes with Gas heating and Ceiling Fans and Central Air cooling. The interior features include 9Ft + Ceiling, Beam Ceiling, Multi Panel Doors and Vaulted Ceiling.


Our Children's Liturgy Programme is a wonderful way for your child to have a meaningful experience at Mass at their own level.
Kinder and primary school aged children are invited to participate each Sunday at the 9.30 am Mass.
The children are divided into two groups. The younger group is for children in pre-School to Year 1 and the older group is for children in Years 2 to 6.
The younger group meets in the chapel and the older group in 'The Meeting Place'. They are instructed in the gospel of the day at a level they can readily understand and participate in an age-appropriate activity. The children love this special time of instruction and the activity which supports their lesson. After the lesson they return to the church where they share what they have learnt with the rest of the congregation. They then join their families in time to participate in the Liturgy of the Eucharist.
Volunteer catechists enthusiastically support this initiative.