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Research ArticleIMMUNOLOGY
De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function
View ORCID ProfileRuohan Wu1,*,
View ORCID ProfileXuyong Chen1,*,
View ORCID ProfileSiwen Kang1,*,
Tingting Wang1,
View ORCID ProfileJN Rashida Gnanaprakasam1,
View ORCID ProfileYufeng Yao2,
Lingling Liu1,
Gaofeng Fan2,
View ORCID ProfileMark R. Burns3 and
View ORCID ProfileRuoning Wang1,†
1Center for Childhood Cancer and Blood Diseases, Hematology/Oncology and BMT, Abigail Wexner Research Institute at Nationwide Children's Hospital, The Ohio State University, Columbus, OH 43205, USA.
2School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
3Aminex Therapeutics Inc., Epsom, NH 03234, USA.
↵†Corresponding author. Email: ruoning.wang{at}nationwidechildrens.org
Science Advances 16 Dec 2020:
Vol. 6, no. 51, eabc4275
DOI: 10.1126/sciadv.abc4275
Ruohan Wu
ORCID record for Ruohan Wu
Xuyong Chen
ORCID record for Xuyong Chen
Siwen Kang
ORCID record for Siwen Kang
Tingting Wang
JN Rashida Gnanaprakasam
ORCID record for JN Rashida Gnanaprakasam
Yufeng Yao
ORCID record for Yufeng Yao
Lingling Liu
Gaofeng Fan
Mark R. Burns
ORCID record for Mark R. Burns
Ruoning Wang
ORCID record for Ruoning Wang
For correspondence: ruoning.wang@nationwidechildrens.org
Robust and effective T cell–mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of biosynthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in extracellular polyamine uptake, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular polyamine pool, inhibits T cell proliferation, and suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine pool for managing inflammatory and autoimmune diseases.
To cope with the capacity of pathogens for exponential growth, T cells have evolved mechanisms for rapidly adjusting their metabolism in response to T cell receptor (TCR) activation and additional signals indicating environmental change. A robust and effective metabolic reprogramming enables T cells to rapidly expand in number and differentiate to achieve large numbers of effector cells specialized in producing high levels of cytokines. Emerging evidence has shown that metabolic rewiring of the central carbon metabolism maximizes the acquisition and assimilation of energy and carbon sources and prepares T cells for growth, differentiation, immune regulation, and defense (1–5). Glycolysis, the pentose phosphate pathway (PPP), the Krebs cycle, and fatty acid oxidation (FAO) represent a core set of metabolic pathways that transform carbon and chemical energy from environmental nutrients to support the bioenergetic and biosynthesis needs of T cells. Beyond that, a myriad of peripheral metabolic pathways are integrated in complex metabolic networks and are tightly regulated to generate specialized metabolites, which are essential for maintaining homeostasis and immune functions of T cells.
Nonessential amino acids including arginine, glutamine, and proline have both anabolic and catabolic functions, providing building blocks for protein and nucleic acids, and connecting central carbon metabolism to a variety of specialized metabolic pathways, including polyamine biosynthesis (6–9). Polyamine is an essential class of polycationic metabolites ubiquitously present in all living organisms. In addition to de novo biosynthesis, other metabolic routes including polyamine catabolism, influx, and efflux act in concert to determine the size of the intracellular polyamine pool (10). Disrupting polyamine homeostasis can affect a plethora of cellular processes, including differentiation, transcription, translation, redox balance, and mitochondria quality control (10). Dysregulation of the level of polyamine and its amino acid precursors has been found to be associated with inflammation and autoimmune diseases (11, 12). We have previously reported that polyamines are one of the most up-regulated metabolite groups following T cell activation, and transcription factor MYC is responsible for its up-regulation (13). Emerging evidence has also shown that polyamine homeostasis is tightly regulated in cellular contexts other than T cells, which have critical roles in immune regulation and defense (14–18). As such, a better understanding of how polyamine homeostasis is regulated in immune cells will reveal the fundamental principles of the emerging connections between immune cell metabolic fitness and functional robustness. Further knowledge will also enable us to devise rational and practical approaches to treat inflammatory and autoimmune diseases.
Intrinsic T cell signaling cascades are instrumental in the control of T cell metabolic programming (5, 19–23). Numerous extrinsic environmental factors including oxygen and nutrient supplies also substantially influence T cell metabolic phenotypes and, thus, immune functions in vivo (24). Here, we report that the intracellular polyamine pool is tightly regulated by de novo biosynthesis and cellular uptake, through the import of extracellular polyamine. Heightened arginine and glutamine catabolism provide carbon sources to support polyamine de novo biosynthesis in vitro. Genetic and pharmacologic ablation of de novo biosynthesis of polyamine is sufficient to deplete the polyamine pool and suppress T cell proliferation in vitro. However, de novo biosynthesis is not essential for driving T cell proliferation in vivo, where T cells can salvage circulating polyamine to maintain intracellular polyamine pool. Simultaneously blocking polyamine synthesis and salvage inhibits T cell proliferation in vivo and confers protection against the pathogenic development of experimental autoimmune encephalomyelitis (EAE). Our findings implicate the potential therapeutic value of targeting polyamine metabolism in treating and managing inflammatory and autoimmune diseases.
Inhibition of ornithine decarboxylase reduces T cell proliferation and viability in vitro
We previously reported that an MYC-dependent noncanonical metabolic pathway links amino acid catabolism to the biosynthesis of polyamines during T cell activation (13). To investigate the role of polyamine metabolism in T cells, we used a genetic and a pharmacologic approach to ablate polyamine de novo biosynthesis. Because ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine de novo biosynthetic pathway, is essential for early embryo development, and ODC germline knockout is embryonically lethal (25), we obtained a mouse strain containing a reporter-tagged conditional allele of ODC (FRT-LacZ; ODCfl) generated by the European Mouse Mutant Archive (26). We first crossed this strain with the flippase (FLP) knock-in mouse strain, which removed the LacZ reporter allele and generated the strain containing the conditional allele (ODCfl). Then, we generated a T cell–specific ODC knockout strain (ODC cKO) by crossing the ODCfl strain with the CD4-Cre strain. The deletion of ODC was validated by quantitative polymerase chain reaction (qPCR) (fig. S1A), and the ablation of polyamine de novo synthesis was further validated by the accumulation of ornithine (substrate of ODC) and the depletion of putrescine, N-acetylputrescine, spermine, and spermidine (fig. S1B). ODC deletion did not affect the distribution of T cell subsets in the thymus, spleen, and lymph nodes (fig. S2, A and B). In addition, the percentage of naturally occurring interferon-γ (IFN-γ)–producing, interleukin-17 (IL-17)–producing, and FoxP3+ CD4 T cells is comparable in both wild-type (WT) and ODC cKO animals (fig. S2C). However, genetic deletion of ODC substantially delayed cell cycle progression from G0/G1 to the S phase after T cell activation and suppressed overall T cell proliferation in vitro (Fig. 1, A and B). Consistent with the impact of genetic deletion of ODC on T cells, difluoromethylornithine (DFMO), a potent inhibitor of ODC, inhibited activation-induced T cell cycle progression and proliferation in vitro (Fig. 1, C and D). Last, both genetic deletion of ODC and DFMO treatment caused moderately more cell death after activation in a time-dependent manner (Fig. 1, E and F). Together, our results indicate that polyamine homeostasis is critical for T cell proliferation and survival.
Fig. 1 Blockage of de novo polyamine biosynthesis suppresses T cell proliferation and reduces viability in vitro.
(A and C) Cell cycle of activated CD4+ T cells at 24, 36, and 48 hours with indicated genotypes or treatments was analyzed by using BrdU and 7AAD staining; the numbers indicate the percentage of cells in each cell cycle stage. Data represent three independent experiments. (B and D) Cell proliferation of naïve and activated CD4+ T cells at 48 and 72 hours with indicated genotypes or treatments was determined by CFSE dilution. Data represent six independent experiments. (E and F) Cell viability of activated CD4+ T cells at 24, 48, and 72 hours with indicated genotypes or treatments was assessed by 7AAD staining. Top panel is the representative dot plot, and bottom panel is the statistical analysis (means ± SEM). FSC, forward scatter. Data represent three independent experiments, **P < 0.01, ***P < 0.001 compared with WT, one-way analysis of variance (ANOVA). n.s., not significant.
ODC activity is dispensable for T cell proliferation and function in vivo
Controlling polyamine homeostasis is crucial for supporting cellular functions in all cell types, and both de novo biosynthesis and salvage by importing extracellular polyamine into the cell tightly regulate cellular polyamine pool size (10). Without exogenous polyamine supplements in cell culture media, we envisioned that T cells would solely depend on de novo biosynthesis to maintain the intracellular polyamine pool in vitro. However, circulating polyamines that are provided by dietary intake and by intestinal microbiota may be a source of exogenous polyamine for T cells in vivo (27). To assess the impact of ablating de novo biosynthesis on CD4 T cell proliferation in vivo, we used a well-established competitive homeostatic proliferation assay to determine the ratio and carboxyfluorescein succinimidyl ester (CFSE) dilution pattern of purified WT(Thy1.1+) or ODC cKO(Thy1.2+) CD4+ T cells in Rag1-deficient (Rag1−/−) mice, which lack endogenous T cells and permit spontaneous proliferation of adoptively transferred donor T cells. Unexpectedly, the ratio between WT and ODC cKO CD4+ T cells was similar before and after adoptive transfer. In addition, WT and ODC cKO CD4+ T cells display an overlapped CFSE dilution pattern, indicating that the loss of ODC did not affect T cell proliferation in vivo (Fig. 2A). Next, we sought to measure antigen-specific, TCR-dependent proliferation of WT or ODC cKO CD4+ T cells. We crossed Thy1.1 and CD4-Cre, ODCfl mice with OT-II transgenic mice to generate WT(Thy1.1+) and ODC cKO(Thy1.2+) donor OT-II strains in CD45.2+ background. We then adoptively transferred mixed and CFSE-labeled WT and ODC cKO CD4+ T cells into CD45.1+ mice that were immunized with chicken ovalbumin 323–339 peptide (OVA323–339) in complete Freund's adjuvant (CFA). After 7 days, we measured the percentage ratio and CFSE dilution pattern of WT(Thy1.1+) and ODC cKO(Thy1.2+) CD4+ T cells in popliteal lymph node. Consistent with the homeostatic proliferation results, WT and ODC cKO OT-II–specific CD4+ T cells display a comparable antigen-specific proliferation (Fig. 2B). The expansion and balance between proinflammatory CD4+ T effector (Teff) cells determine the pathogenic development of EAE, a murine model of multiple sclerosis (MS), which is an inflammatory demyelinating disease of the central nervous system (CNS). We used this well-characterized system to further interrogate an in vivo CD4 T cell response in the absence of polyamine de novo biosynthesis. In line with our homeostatic and antigen-specific proliferation data, neither the genetic deletion of ODC in T cells nor the systemic delivery of DFMO changes the kinetics of pathogenic progression (Fig. 2, C and D). Together, our data indicate that polyamine salvage from circulation may be able to support T cell proliferation and effector function by compensating for the loss of de novo biosynthesis in the in vivo environment.
Fig. 2 De novo polyamine biosynthesis is dispensable for driving T cell proliferation and function in vivo.
(A and B) An overview of in vivo competitive proliferation experimental procedure. The donor CD4 T cell ratios before and after adoptive transfer were evaluated by surface staining of isogenic markers, and cell proliferation was evaluated by CFSE dilution. Data represent three independent experiments. i.v., intravenous. (C and D) An overview of EAE experimental procedure. Clinical scores were evaluated daily (right). Data represent means ± SEM (n = 5) for each group; experiments were repeated three times.
Polyamine salvage compensates for the loss of biosynthesis activity in vitro
Next, we investigated the role of polyamine salvage (uptake) in regulating polyamine homeostasis in T cell. We measured polyamine uptake activity using radioactive-labeled putrescine (14C-putrescine) in naïve, active WT and ODC cKO T cells. Active T cells displayed higher polyamine uptake activity than naïve T cells (Fig. 3A). Ablation of ODC induces a compensatory increase in polyamine uptake (Fig. 3B). Next, we asked if polyamine uptake is sufficient to maintain polyamine homeostasis and support T cell proliferation in the absence of ODC activity. While genetic deletion or pharmacologic inhibition of ODC substantially delayed cell cycle progression from G0/G1 to the S phase and suppressed overall T cell proliferation, exogenous polyamine supplement could restore the cell cycle progression, proliferation, and viability in DFMO-treated and ODC cKO CD4+ T cells in vitro (Fig. 3, C and F, and fig. S3, A and B). We then sought to use a pharmacologic approach to block polyamine uptake and assessed the role of polyamine uptake in regulating polyamine homeostasis. AMXT 1501 (AMXT) is a novel lipophilic polyamine mimetic that potently blocks polyamine uptake in the low nanomolar concentration (28). The combination of AMXT and DFMO could effectively deplete the polyamine pool in tumor cells and suppress the growth of tumors (29). These promising preclinical studies led to a recently opened phase I clinical trial in solid tumors (NCT03077477). Similar to the genetic data (Fig. 3B), DFMO treatment induces a compensatory increase in polyamine uptake, which can be blocked by AMXT (Fig. 3G). In addition, AMXT could significantly suppresses exogenous polyamine-mediated cell proliferation and viability in ODC cKO, but not in WT CD4+ T cells (Fig. 3H and fig. S3C). Similarly, DFMO treatment suppresses human T cell viability and proliferation, which can be restored by polyamine supplement (fig. S4, A and B). AMXT treatment blocks exogenous polyamine-mediated cell proliferation and viability in human T cells (fig. S4, A and B). These results demonstrated that both human and mouse T cells require polyamines for supporting proliferation and survival. In addition, our results suggest that either polyamine biosynthesis or salvage is sufficient to maintain polyamine homeostasis in T cells. Supporting this idea, AMXT treatment alone failed to suppress T cell homeostatic proliferation or antigen-specific proliferation in vivo (fig. S5, A and B). These results, together with the results described above, suggest that the salvage pathway and the de novo biosynthesis pathway can compensate for the loss of each other, representing a layer of metabolic plasticity engaged by T cells to maintain polyamine homeostasis.
Fig. 3 Ablation of de novo polyamine biosynthesis renders T cells dependent on polyamine uptake that can be blocked by AMXT 1501.
(A, B, and G) Putrescine uptake in the indicated experimental groups was determined by the cellular retention of 14C-labeled putrescine (means ± SEM). Data represent three independent experiments. ***P < 0.001, Student's t test. (C and E) Cell cycle of 48-hour–activated CD4+ T cells with indicated genotypes or treatments was assessed by using BrdU and 7AAD staining. The numbers indicate the percentage of cells in each cell cycle stage. Data represent three independent experiments. (D, F, and H) Cell proliferation of activated CD4+ T cells at 48 and 72 hours with indicated genotypes or treatments was evaluated by CFSE dilution. Data represent three independent experiments.
Simultaneously blocking polyamine salvage and biosynthesis suppresses T cell proliferation in vivo
Given that either genetically ablating ODC or pharmacologically blocking polyamine uptake failed to affect T cell proliferation and function in vivo, we sought to assess the impacts of simultaneously blocking polyamine salvage and biosynthesis on T cells in vivo. We used competitive homeostatic proliferation and antigen-specific T cell proliferation assays to determine the proliferation of WT and ODC cKO donor CD4+ T cells in recipient mice, which were treated with vehicle or AMXT during the course of experiment. Donor ODC cKO CD4+ T cells recovered from AMXT-treated animals but not from vehicle-treated animals displayed reduced percentage and delayed proliferation compared with competitive WT CD4+ T cells (Fig. 4, A and B). We then sought to assess the impacts of simultaneously blocking polyamine salvage and biosynthesis on T cells in the EAE model. The genetic deletion of ODC in T cells failed to cause any notable changes in EAE pathogenic progression. Animals that were treated with AMXT displayed a delayed disease onset initially, but eventually proceeded with pathologic development and reached the end point. The combination of AMXT with genetic deletion of ODC in T cells conferred full protection against EAE pathogenic progression (Fig. 4C). ODC inhibitor DFMO is a Food and Drug Administration–approved medicine for hirsutism and African sleeping sickness and has been widely tested as a chemopreventive and chemotherapeutic agent against solid tumors (30, 31). Similar to the genetic data, DFMO alone failed to suppress EAE pathogenic progression (Fig. 4D). Although AMXT alone was sufficient to delay EAE onset moderately, it failed to protect animals from reaching the end point (Fig. 4D). We envision that the combination of DFMO and AMXT may be sufficient to deplete the T cell polyamine pool and consequently suppress T cell proliferation and effector function in vivo. Supporting this idea, the combination of AMXT and DFMO, but not single treatments, conferred full protection against EAE pathogenic progression (Fig. 4D). To evaluate the potential hematological toxicity of DFMO and AMXT combination treatment, we treated CD-1 mice with AMXT or AMXT plus DFMO at various dose levels for 28 days, and then evaluated a range of hematological parameters at days 29 and 43 (after 15-day recovery period). Our results showed that AMXT and DFMO combination treatment does not affect most of the hematological parameters (table S1). Inflammatory TH1, TH17, and FoxP3-expressing regulatory T cells (Tregs) are closely related to CD4 T cell subsets but with distinct functions. The balance between proinflammatory CD4+ Teff cells and Tregs determines the pathogenic development of EAE. Next, we examined polyamine's role in CD4 Teff cell differentiation in vitro. Without exogenous polyamine supplemented in cell culture media, maintenance of the intracellular polyamine pool depends solely on ODC-mediated polyamine biosynthesis. Intracellular polyamine depletion resulting from ODC deficiency inhibited proinflammatory TH1 and TH17 cell differentiation while enhancing anti-inflammatory iTreg cell differentiation in vitro (fig. S6A). In EAE animals treated with DFMO in combination with AMXT, we observed a significant reduction in CD4+ T cell infiltration into the CNS and a reduction in IL17+ CD4+ T cell in CNS compared with the control group (fig. S6B). Together, our results indicate that the polyamine blocking approach, via ablation of salvage and biosynthesis pathways, suppresses T cell proliferation and inflammatory functions and may be a potential new therapy for treating inflammatory and autoimmune disease.
Fig. 4 Simultaneous blockage of polyamine uptake and de novo biosynthesis suppresses T cell proliferation and function in vivo.
(A and B) Overview of in vivo competitive homeostatic and antigen-driven proliferation experimental procedure. The donor CD4+ T cell ratios before and after adoptive transfer were evaluated by surface staining of isogenic markers, and cell proliferation was evaluated by CFSE dilution. Data represent three independent experiments. s.c., subcutaneous. (C and D) Overview of the EAE experimental procedure. Clinical scores were evaluated daily. Data represent means ± SEM of n = 5 to 10 for each group. Experiments were repeated three times. **P < 0.01, ***P < 0.001, two-way analysis of variance (ANOVA).
Amino acid catabolism provides carbon sources for polyamine biosynthesis
Dietary intake and intestinal microbiota metabolism are major sources of circulating polyamine (27). We reasoned that an understanding of carbon sources that drive polyamine biosynthesis in T cells would enable the development of nutritional approaches for effectively controlling polyamine homeostasis in T cells. While arginine catabolism is integrated into the urea cycle to detoxify ammonia and provides the precursor for polyamine biosynthesis, we previously reported that glutamine-derived carbon could be funneled into polyamine biosynthesis through ornithine in T cells (13). In addition, proline can also provide carbon for polyamine biosynthesis in the placenta and in plants (32). To determine to what extent these three amino acids contribute to polyamine biosynthesis, we applied the stable isotope of carbon-13 (13C) labeling and mass spectrometry approach. We supplied 13C6-arginine, 13C5-glutamine, or 13C5-proline as metabolic tracers in T cell culture media and then followed 13C incorporation into individual metabolites (Fig. 5A). While not all selected metabolites were detected in our experiment because of technical limitation and a portion of proline was produced through de novo biosynthesis, our results clearly demonstrated that 13C5-proline only contributes a minimal amount of 13C5 isotopologues of ornithine and polyamine (Fig. 5B). In contrast, 13C6-arginine and 13C5-glutamine contribute 50 and 40% of 13C5-ornithine, respectively (Fig. 5B). 13C6-arginine and 13C5-glutamine contribute around 80 and 20% of 13C4 isotopologues of polyamine (putrescine and spermidine generated via decarboxylation of ornithine), respectively (Fig. 5B). Thus, we conclude that arginine is a major carbon donor, and glutamine is a minor carbon donor for supporting polyamine biosynthesis in T cells in vitro.
Fig. 5 Arginine catabolism supports polyamine biosynthesis and T cell proliferation.
(A) Diagram of putative catabolic routes of 13C5-glutamine, 13C6-arginine, and 13C5-proline in T cells. (B) T cells were incubated in 13C5-glutamine or13C6-arginine, or 13C5-proline medium for 36 hours. Metabolites were extracted and analyzed using CE-TOFMS as described in Materials and Methods. Numbers in the x axis represent those of 13C atoms in given metabolites. Data represent means ± SEM of n = 3 for each group. (C) Cell viability of activated CD4+ T cell with indicated treatments was determined by 7AAD staining. Data represent three independent experiments. (D and E) CD4+ T cells were activated and polarized toward iTreg, TH1, and TH17 lineages with indicated treatment for 72 hours. The indicated proteins were quantified by intracellular staining. Cell proliferation was determined by CFSE staining. Data represent three independent experiments, ***P < 0.001, ****P < 0.0001, one-way ANOVA.
Arginine catabolism drives T cell proliferation partially through supporting polyamine biosynthesis
While arginine is generally considered a nonessential amino acid, T cells are arginine auxotrophic in vitro (18). Arginine is required to maintain CD4+ T cell viability, driving proliferative and proinflammatory lineage (TH17) differentiation (Fig. 5, C to E). While the amount of most amino acids is in the low micromolar (μM) range, the concentration of arginine is in the millimolar (mM) range in cell culture media. We envisioned that, in addition to a general requirement of arginine for protein synthesis, arginine might also support T cell proliferation and differentiation through polyamine biosynthesis. To test this idea, we titrated down the amount of arginine in cell culture media and found that 10 μM arginine was sufficient to support protein synthesis and the growth of cell mass at the early time point after T cell activation but failed to maintain cell viability and drive proliferation later (fig. S7, A and B). We envision that arginine supports protein synthesis and polyamine biosynthesis, both of which are required to drive T cell proliferation. Supporting this idea, we have shown that a low level of arginine (10 μM) in culture media is sufficient to support protein synthesis, but not proliferation (fig. S7A). Similar to what we found in arginine depletion condition, a low level of arginine (10 μM, with or without daily replenishment) in culture media failed to support T cell proliferation and an optimal TH17 differentiation (fig. S8, B and C). However, polyamine supplements can partially restore T cell proliferation and TH17 differentiation with a low level of arginine (10 μM, with or without daily replenishment) (fig. S8, B and C). In contrast, polyamine supplements failed to restore T cell proliferation and differentiation under arginine starvation condition (0 μM) (fig. S8A). Collectively, our data indicate that T cell activation engages a metabolic axis that connects arginine catabolism to polyamine de novo biosynthesis.
Fast-growing pathogens impose selective pressures on the metabolic fitness and metabolic plasticity of host immune cells, which are necessary for immune cells to maintain homeostasis while remaining ready to mount rapid responses under diverse metabolic and immune conditions (24, 33–36). A robust T cell–mediated adaptive immune response exhibits high and dynamic metabolic demands in T cells, which is accommodated through fine-tuned regulation on both the central carbon metabolic pathways and peripheral metabolic pathways including polyamine metabolism (4, 5, 9, 19). The standard formulation of cell culture media, which does not fully recapitulate the physiological metabolite composition in the plasma and tissue microenvironment, often leads to a substantial metabolic adaptation of growing cells in vitro. Metabolic phenotypes of cells growing in vivo may further differ from cells growing in vitro as a result of other environmental factors including oxygen level, cellular competition and cooperation, and the biophysical properties of the extracellular matrix (37–39). Similarly, T cell metabolism can respond and adapt to environmental nutrient levels (24, 40). Our studies revealed T cells' capacity to engage in both de novo biosynthetic and salvage pathways to fine-tune polyamine homeostasis, which is required to maximize metabolic fitness and optimize CD4 Teff cell proliferation and differentiation. Such metabolic plasticity is likely to be crucial for T cells' ability to elicit robust immune responses in different tissue contexts.
Glutamine and arginine are two nonessential amino acids that not only fulfill the general requirements for protein synthesis but also connect central carbon metabolism to a variety of biosynthetic pathways to produce specialized metabolites (41–44). Glutamine catabolism funnels the anaplerotic flux of carbon into the TCA cycle and also provides sources of nitrogen and carbon to support biosynthesis of nonessential amino acids, lipids, nucleotides, glutathione, and polyamines in T cells (13, 45, 46). Similarly, arginine is critical for maintaining T cell viability, driving proliferative and effector functions (14). We found that polyamine supplement could partially relieve T cell's dependence on arginine, indicating that a key role of arginine catabolism in T cells is to support polyamine biosynthesis. Consistently, arginine serves as a major donor of carbon in polyamine biosynthesis, while glutamine only plays a minor role in funneling carbon into polyamine in the presence of arginine in vitro. These two amino acids contribute a comparable portion of carbon to ornithine—the precursor of polyamine. This finding is in line with previous studies showing that arginine-derived ornithine is not the only source for endogenous ornithine in most mammalian tissues (47). In addition, our findings may implicate ornithine as an important metabolic node representing a key branch point in both glutamine and arginine catabolic pathways. Ornithine can be committed toward the urea cycle, proline biosynthesis, or de novo synthesis of polyamine. The production of ornithine from two different metabolic precursors, glutamine and arginine, also enables fine-tuned coordination between the metabolic flux shunted toward polyamine synthesis and the metabolic flux shunted toward other specialized metabolites. Consistent with this idea, the overall high consumption rate of glutamine and arginine may provide a sensitive and precise regulation on intermediate metabolites that can be committed toward several metabolic branches, hence permitting rapid responses to meet the metabolic demands of cell growth and cytokine production.
Active T cells and other highly proliferative cells such as tumor cells share metabolic characteristics and are strictly dependent on the catabolism of glucose and glutamine through the central carbon metabolic pathways (48). Similarly, elevated levels of polyamine are associated with T cell activation and cell transformation (13, 49). Pharmacologic or genetic targeting of ODC, a transcriptional target of proto-oncogene MYC, could delay the development and progression of MYC-driven tumors in mice (50). While pharmacologic agents targeting polyamines such as the ODC inhibitor DFMO have generally low toxicity, DFMO yields only very marginal therapeutic benefits in patients with cancer as a single-agent therapy in clinical trials (51). Clearly, cancer cells are capable of importing polyamine from circulation to overcome the effect of DFMO. Supporting this idea, the simultaneous blockage of polyamine biosynthesis and uptake has generated promising results in tumor preclinical animal models (29). Mammalian cells can uptake polyamine through endocytosis and membrane transport system mediated by several solute carrier transporters (52). AMXT 1501 is a novel lipophilic polyamine mimetic that potently blocks polyamine uptake through competing with polyamine (28). The combination of AMXT 1501 and DFMO could effectively deplete the polyamine pool in tumor cells and suppress the growth of tumors in various animal models (29). These promising preclinical studies led to a recently opened phase I clinical trial in solid tumors (NCT03077477). Similarly, we have shown that ODC cKO CD4+ T cells proliferate and function normally in vivo. However, concurrent treatment of ODC cKO CD4+ T cells with AMXT 1501 abrogates their proliferation and inflammatory function in vivo. Moreover, the combination of AMXT 1501 and DFMO could confer full protection to mice against pathogenic development of EAE. Thus, polyamine blocking strategy via simultaneous blockade of polyamine biosynthesis and salvage may present a promising and novel therapy for treating inflammatory and autoimmune diseases.
C57BL/6 (WT) mice, Rag1−/− mice (B6.129S7-Rag1tm1Mom/J), OT-II mice [B6.Cg-Tg(TcraTcrb)425Cbn/J], Thy1.1+ mice (B6.PL-Thy1a/CyJ), and CD45.1+ mice (B6.SJL-Ptprca Pepcb/BoyJ) were obtained from the Jackson laboratory (JAX, Bar Harbor, ME). Mice with one targeted allele of ODC on the C57BL/6 background (Odc1tm1a(EUCOMM)Hmgu/J) were generated by the Knockout Mouse Phenotyping Program (KOMP2) at the JAX. The mice were first crossed with a transgenic Flippase strain [B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ] to remove the LacZ reporter allele, and then crossed with the CD4-Cre strain to generate T cell–specific ODC knockout strain (ODC cKO). Gender- and age-matched mice (6 to 12 weeks old) were used for all the experiments. All mice were maintained in specific pathogen–free conditions at the Nationwide Children Hospital animal facility, and all the animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Abigail Wexner Research Institute at Nationwide Children's Hospital (protocol number AR13-00055).
For analysis of surface markers, cells were stained in phosphate-buffered saline (PBS) containing 2% (w/v) bovine serum albumin and the appropriate antibodies from BioLegend for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated for 4 hours with Cell Stimulation Cocktails of phorbol 12-myristate 13-acetate (PMA), ionomycin, brefieldin A, and monensin (eBioscience). Cells were then washed with PBS, fixed, and permeabilized using FoxP3 Fixation/Permeabilization Kit according to the manufacturer's instructions (eBioscience). Cells were then stained with anti–IFN-γ and anti–IL-17A antibodies (BioLegend); for intracellular FoxP3 staining, cells were fixed, permeabilized, and stained with anti–FoxP3 antibody (BioLegend). For protein content measurement, cells were fixed with 4% paraformaldehyde for 30 min at 4°C, permeabilized with FoxP3 permeabilization solution (eBioscience), and then stained with fluorescein isothiocyanate (FITC) (Thermo Fisher Scientific). For CFSE staining, freshly isolated T cells were incubated with 4 μM CFSE (Invitrogen) in PBS at room temperature for 10 min, and then washed three times with cell culture medium containing 10% fetal bovine serum (FBS). For cell viability analysis, cells were stained with 7AAD (BioLegend) for 5 min before running on a flow cytometer. All flow cytometry data were acquired on Novocyte (ACEA Biosciences) and were analyzed with FlowJo software (BD Biosciences).
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat (American Red Cross) using Ficoll/Hypaque density gradient centrifugation. Human CD4+ naïve T cells were directly enriched from PBMCs using human CD4 naïve T cell Isolation Kit (MojoSort, BioLegend) following the manufacturer's instructions. Isolated human T cells (1 × 106 cells/ml) were stimulated with plate-bound anti-hCD3 and anti-hCD28 antibodies and cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated dialyzed FBS (DFBS), 2 mM l-glutamine, 1% sodium pyruvate (Sigma-Aldrich), penicillin (100 U/ml), and streptomycin (100 μg/ml), in the presence of hIL-2 (100 U/ml, Bio X Cell) at 37°C/5% CO2. Culture plates were precoated with anti-hCD3 (1 μg/ml) and anti–hCD28 antibodies (1 μg/ml) (Bio X Cell) overnight at 4°C. DFBS was made by dialyzing against 100 volumes of PBS (five changes in 3 days) using Slide-A-Lyzer G2 dialysis cassettes with cut-through MW size 2K (Thermo Fisher Scientific) at 4°C to remove any potential polyamines in the FBS (Atlanta Biologicals).
Total CD3+ T cells or naïve CD4+ T cells were isolated from mouse spleen and lymph nodes using murine CD3 or CD4 naïve T cell Isolation Kit (MojoSort, BioLegend) following the manufacturer's instructions. Freshly isolated mouse T cells (1 × 106 cells/ml) were either maintained in culture media containing mouse IL-7 (5 ng/ml) or stimulated with mouse IL-2 (100 U/ml) as well as plate-bound anti-mCD3 and anti-mCD28 antibodies, and cultured with the medium described above with 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). Culture plates were precoated with anti-mCD3 (2 μg/ml) (clone 145-2C11, Bio X Cell) and anti-mCD28 (2 μg/ml) (clone 37.51, Bio X Cell) antibodies overnight at 4°C. For CD4 T cell polarization, freshly isolated naïve CD4+ T cells (0.6 × 106/ml) were stimulated with cytokine and neutralizing antibody cocktail as well as plate-bound anti-mCD3 and anti-mCD28 antibodies. Specifically, culture plates were precoated with 5 μg/ml (for iTreg and TH1) or 10 μg/ml (for TH17) anti-mCD3 and anti-mCD28 antibodies overnight at 4°C. Cytokines and neutralizing antibody cocktails include mIL-2 (200 U/ml) and hTGF-β1 (5 ng/ml, PeproTech) for iTreg differentiation; mIL-6 (50 ng/ml, PeproTech), hTGF-β1 (2 ng/ml), anti–mIL-2 (8 μg/ml, Bio X Cell), anti–mIL-4 (8 μg/ml, Bio X Cell), and anti–mIFN-γ (8 μg/ml, Bio X Cell) for TH17 differentiation; and mIL-2 (200 U/ml), mIL-12 (5 ng/ml, PeproTech), and anti–mIL-4 (10 μg/ml) for TH1 differentiation. DFMO (2 mM, Carbosynth) and AMXT 1501 (1 μM, Aminex Therapeutics) were added into the cell culture medium in the indicated experiments. For polyamine supplementation experiment, polyamine mixture including putrescine, spermidine, and spermine (1 μM, Sigma-Aldrich) was added into culture medium along with aminoguanidine (0.2 mM, Sigma-Aldrich), which inhibits diamine oxidase in the FBS.
Cell cycle analysis
T cell cycle analysis was performed using Phase-Flow Alexa Fluor 647 BrdU Kit (BioLegend) per the manufacturer's instructions. Briefly, T cells were pulsed with BrdU (5-bromo-2′-deoxyuridine) (10 μg/ml) for 1 hour followed by surface staining, fixation, and permeabilization. BrdU incorporated into the DNA during the S phase was recognized by intracellular BrdU antibody staining, and total DNA content was used to differentiate the G1 and G2 stages by 7AAD labeling.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was reverse transcribed using random hexamers and M-MLV Reverse Transcriptase (Invitrogen). SYBR Green–based reverse transcription qPCR (RT-qPCR) was performed using the Bio-Rad CFX96 Real-Time PCR Detection System. The relative gene expression was determined by the comparative CT method, also referred to as the 2−ΔΔCT method. The data were presented as the fold change in gene expression normalized to an internal reference gene (β2-microglobulin) and relative to the control (the first sample in the group). Fold change = 2−ΔΔCT = [(CTgene of interest − CTinternal reference)] sample A − [(CTgene of interest − CTinternal reference)] sample B. Samples for each experimental condition were run in triplicated PCRs. ODC1 primer sequences (forward: GCATGCATCTGCTTGATATTGG, reverse: GTCCAGAGCTGGGTTGATTAC) were obtained from PrimerBank.
Putrescine uptake assay
T cells (2 × 106) were suspended in 200 μl of PBS containing permeant putrescine (100 μM) and [1,4-14C]-putrescine dihydrochloride (0.2 μCi, ARC 0245) and then incubated at 37°C for 10 min (within the established linear phase of uptake). The uptake was stopped by loading all the transport mixture onto a discontinuous gradient of bromododecane and perchloric acid/sucrose and then centrifuged at 14, 000g for 90 s. The discontinuous gradient was prepared by overlaying 1-bromododecane (800 μl, Sigma-Aldrich) above 20% perchloric acid (100 μl, Sigma-Aldrich)/8% sucrose solution in a 1.5-ml microfuge tube. The samples were snap frozen in an ethanol-dry ice bath. The bottom part of the microfuge tubes containing T cell lysate in perchloric acid–sucrose was cut by a microfuge cutter. The cell lysates were diluted with 300 μl of 0.5% SDS–1% Triton X-100, and then transferred into scintillation vials with 10 ml of scintillation cocktail. The radioactivity was quantitated by liquid scintillation spectrometry.
Adoptive cell transfer and in vivo proliferation
For homeostatic proliferation assay, naïve CD4+ T cells isolated from WT(Thy1.1+) and ODC cKO(Thy1.2+) donor mice were labeled with CFSE as described above, and then were mixed at 1:1 ratio. Approximately 1 × 107 mixed cells in 150 μl of PBS were injected via the retro-orbital venous sinus into gender-matched Rag1−/− mice, which were euthanized 4 days later. Lymph nodes were collected, and cell proliferation was assessed by flow cytometry analysis.
For OVA antigen–driven proliferation assay, naïve CD4+ T cells isolated from OT-II–WT(Thy1.1+) and OT-II–ODC cKO(Thy1.2+) donor mice were labeled with CFSE as described above, and then were mixed at 1:1 ratio. Approximately 1 × 107 mixed cells in 150 μl of PBS were injected via the retro-orbital venous sinus into gender-matched CD45.1+ mice. One day after cell injection, host mice were immunized subcutaneously in the hock area of both legs with 50 μl OVA323–339 peptide (1 mg/ml, InvivoGen) emulsified with CFA (InvivoGen). Seven days after immunization, lymph organs of host mice were collected for cell surface staining and flow cytometry analysis.
EAE induction and assessment
Mice were immunized subcutaneously with 100 μg of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide emulsified in CFA [mixture of IFA (incomplete Freund's adjuvant; Difco) and Mycobacterium tuberculosis (Difco)]. Mice were intraperitoneally injected with 200 ng of pertussis toxin (PTX; List Biological Laboratories) on the day of immunization and 2 days later. For the DFMO-treated group, mice were fed with 1% DFMO in drinking water, which was replenished every 5 days throughout the experiment. For the AMXT 1501–treated group, mice received AMXT 1501 (3 mg/kg) subcutaneously daily throughout the experiment. All mice were observed daily for clinical signs and scored as described below in Table 1.
Table 1 EAE clinical score assessment sheet.
In some experiments, mice were euthanized when control mice reached the onset of symptom or when control mice reached terminal end stage as indicated. The CNS (brain and spinal cord), spleen, and peripheral lymph nodes were collected, and then single-cell suspension of each organ was prepared. To isolate mononuclear cells from the CNS, cell suspension was centrifuged on a 30%/70% Percoll gradient at 500g for 30 min. Isolated cells were activated for 4 hours using cell stimulation cocktail, and surface staining and intracellular staining were performed before flow cytometric analysis as described above.
Capillary electrophoresis–triple quadrupole/time-of-flight mass spectrometry (CE-QqQ/TOFMS) analysis
CD3+ T cells (around 1.5 × 107 cells per sample) isolated from WT or ODC cKO mice were activated for 36 hours and then used for the extraction of intracellular metabolites. The cells were collected by centrifugation (300g at 4°C for 5 min) and washed twice with 5% mannitol solution (10 ml first and then 2 ml), then treated with 800 μl of methanol and vortexed for 30 s to inactivate enzymes. Next, the cell extract was treated with 550 μl of Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies Inc., Tsuruoka, Japan) and vortexed for 30 s. The extract was obtained and centrifuged at 2300g and 4°C for 5 min, and then 700 μl of upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9100g and 4°C for 180 min to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 μl of Milli-Q water for CE-MS analysis. Cationic compounds were measured in the positive mode of CE-TOFMS, and anionic compounds were measured in the positive and negative modes of CE-MS/MS. Peaks detected by CE-TOFMS and CE-MS/MS were extracted using an automatic integration software (MasterHands, Keio University, Tsuruoka, Japan, and MassHunter Quantitative Analysis B.04.00, Agilent Technologies, Santa Clara, CA, USA, respectively) to obtain peak information including m/z (mass/charge ratio), migration time (MT), and peak area. The peaks were annotated with putative metabolites from the Human Metabolome Technologies (HMT) metabolite database based on their MTs in CE and m/z values determined by TOFMS. The tolerance range for the peak annotation was configured at ±0.5 min for MT and ± 10 ppm for m/z. In addition, concentrations of metabolites were calculated by normalizing the peak area of each metabolite with respect to the area of the internal standard and by using standard curves, which were obtained by three-point calibrations.
Hierarchical cluster analysis and principal components analysis were performed by our proprietary software PeakStat and SampleStat, respectively. Detected metabolites were plotted on metabolic pathway maps using VANTED (Visualization and Analysis of Networks containing Experimental Data) software.
CE-TOFMS analysis
CD3+ T cells (around 1.8 × 107 cells per sample) were activated for 36 hours in conditional media (RPMI-1640) containing 2 mM 13C5-glutamine, 1.15 mM 13C6-arginine, or 0.17 mM 13C5-proline, and then used for the extraction of intracellular metabolites as described above. Metabolome measurements were carried out through a facility service at Human Metabolome Technologies Inc. (Tsuruoka, Japan). CE-TOFMS measurement was carried out using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany). The metabolites were analyzed by using a fused silica capillary [50 μm internal diameter (i.d.)× 80 cm total length], with commercial electrophoresis buffer (solution ID: H3301-1001 for cation analysis and H3302-1021 for anion analysis, Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nl) in the cation analysis and 25 s (approximately 25 nl) in the anion analysis. The spectrometer was scanned from m/z 50 to 1000.
Statistical analysis was conducted using the GraphPad Prism software (GraphPad Software Inc.). P values were calculated with two-way analysis of variance (ANOVA) for the EAE experiments. Unpaired two-tailed Student's t test, multiple comparisons of one- or two-way ANOVA, or Dunnett's least significant difference (LSD) test was used to assess differences in other experiments. P values less than 0.05 were considered significant, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/51/eabc4275/DC1
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Acknowledgments: We thank J. Sherman and H. Rodgers for critically reading and editing the manuscript. We thank Dr. Song Guo Zheng for insight and valuable discussions. Funding: This work was supported by 1R21CA227926-01A1 and 1UO1CA232488-01 from the National Institutes of Health (Cancer Moonshot program), 1R01AI114581 from the National Institutes of Health, V2014-001 from the V-Foundation, and 128436-RSG-15-180-01-LIB from the American Cancer Society (to R.W.). Author contributions: R. Wang conceptualized and supervised this work. R. Wu, X.C., and S.K. carried out the experiments. T.W., J.R.G., Y.Y., and L.L. were involved in data collection, analysis, and review. G.F. and M.R.B. provided conceptual input into the study development. M.R.B. provided experimental material and conceptual input into the study development. R. Wu, X.C., S.K., and R. Wang wrote the manuscript. All authors discussed the results and provided feedback on the manuscript. Competing interests: M.R.B., currently, is the president and CSO of Aminex Therapeutics Inc., which is a pharmaceutical start-up company that actively develops polyamine blocking agents. All other authors declare that they have no competing interest. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
Vol 6, No. 51
You are going to email the following De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function
By Ruohan Wu, Xuyong Chen, Siwen Kang, Tingting Wang, JN Rashida Gnanaprakasam, Yufeng Yao, Lingling Liu, Gaofeng Fan, Mark R. Burns, Ruoning Wang
Science Advances 16 Dec 2020 : eabc4275
Polyamine homeostasis determines T cell function.


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Home » News » Spirits news » Whisky news
Kilchoman completes Islay distillery expansion
By Shay Waterworth
Kilchoman Islay single malt whisky distillery has completed the expansion of its distillery which has the potential to double capacity.
A new building has been constructed containing two new stills, a new mashtun and six additional washbacks which will boost capacity to 480,000 litres of alcohol.
"The stillhouse extension is the latest phase of an expansion that started last year with the completion of our new malt floor and kiln, we hope to complete the final phase; a new visitor centre, shop and café within the next 3-4 months," said Kilchoman founder Anthony Wills.
Kilchoman became the first whisky distillery to be built in Islay for 124 years when it was erected in 2005 and is one of the few independent scotch whisky producers.
It remains the only Islay single malt whisky to be completely made on site and from harvesting barley to bottling its whisky.
A post shared by Kilchoman Distillery (@kilchomanwhisky) on Aug 17, 2019 at 4:46am PDT
Wills added: "At a time when I thought I might take a step back we've invested around £6m in upgrades. That said, without an increase in capacity we would be heading towards a situation where all Kilchoman would be sold purely on allocation.
"The investment amounts to almost an entirely new distillery in itself however we wanted to ensure that the new equipment was a mirror image of the original stillhouse and we've been delighted with the quality of the spirit, with no discernible difference in the character of that produced from the new compared to old stills.
"The new equipment will also allow us to experiment more during the malting and peat phases as well as in the stillhouse with yeast varieties and isolating specific spirit runs."
Keywords: Kilchoman distillery
Kilchoman relases this year's Machir Bay

redpajama_set_name: RedPajamaCommonCrawl


The Morenci boys and girls track teams took first place in its home tri meet Wednesday. In the girls meet, Morenci finished with 65 points, Hudson 62 and Whiteford 40.
Libby Rorick won both the long jump (13-9.5) and 3200 (14:26.9). Sabrina Cately took first in the discus (76-11), Bradey Osborn won the 100 hurdles (17.8) and 300 hurdles (55.6), Trinity Kaufman won the 1600 (6:11.8) and 800 (2:51.2). Morenci also took the 1600 and 3200 relays.
For Hudson, Emily Czeiszperger won the pole vault (7-0) and Madison Martinez took the 400 (1:08.9). Hudson's relay winners included the 800 and 400 relays.
In the boys meet, Morenci finished with 80 points, Whiteford 53 and Hudson 42.
Morenci winners included Breckin Alcock in the high jump (5-8) and 110 hurdles (17.9), Rodney Zimmerman in the long jump (19-5.25), Connor Stowell in the 1600 (5:23), and Brennan Osborn in the 3200 (12:54.5). Morenci also won the 3200 relay and 400 relay.
Hudson took the 1600 relay and had the pole vault winner in Tyler Bolenbaugh (10-6) as well as Adya in the 800 (2:29.2) and Devin Decaire in the 200 (24.2).
Dundee, Summerfield at Addison: The Panthers boys and girls track teams went 3-1 combined in their meet against the visiting Vikings and Bulldogs.
The Addison boys defeated Dundee 71.3-65.66 and Summerfield 119-18, while the Panther girls beat the Bulldogs 105-27 but fell to the Vikings 81-56.
Josh Brown led a 1-2 finish for Addison in the 100-meter dash, finishing in 11.96 seconds, ahead of Dominic Young (12.52). Brown also was the leadoff man for a pair of winning relays, as along with Travis Whitaker, Caleb Gramm and Young, that quartet won the 4x100 (46.16) and 4x200 (one minute, 36.74 seconds).
Gramm won the 300 hurdles (42.99) ahead of Nathan Rodlund (45.01) while Rodlund won the 110 hurdles (16.37) in another 1-2 ahead of Noah Hermansen (16.45). Zach Morse, Matt Poling, Zac Steiner and Gramm took first in the 4x400 with a 3:42.92.
Marquis Bills and Matt Sylvester each won a field event for the boys, with Bills winning the high jump (five feet, 10 1/2 inches) and Sylvester the long jump (personal-best 20'0").
Junior Ally McLouth won three events for the girls, taking the top step in the 100 (17.18) and 300 hurdles (49.17), along with the long jump (15' 4 3/4"), and was also the closing runner for the 4x400 team of Courtney Flaws, Emily Knight and Emily Bayes (4:37.55).
Alexis Chapman won the 100 dash (14.19), and was the closing runner of the relay team of Alivia Magnus, Gracie Worsham and Flaws that won the 4x200 (2:00.45), with Adrienne Corbin rounding out the Panther winners with a win in the discus (97'9").
Blissfield at Summerfield: At Petersburg, the Royals swept Summerfield, 14-3 and 12-7.
Alexa Mapstone and Karis Weirich were the winning pitchers for the 2-0 Royals.
In the first game, Mapstone went four innings, allowing just one hit and striking out three. Makenna Ricker and Cendell Burgermeister had three hits each and Courtney Mason went 2-for-4 with three RBIs. Emily Smith, Emma Vanburen and Mapstone also had multiple-hit games.
In Game 2, Abby Fisher had two hits, including a homerun, and two RBIs. Weirich struck out three in her two innings of work in getting the win.
Homer at Sand Creek: Sand Creek will take a two-game winning streak into spring break after sweeping Homer, 8-4, 9-0.
In the opener, Hannah Reckner went seven innings, allowing seven hits and striking out eight. At the plate, she hit a home run and had two RBIs. Freshman Jerzee Still had a double in her debut. Rylee Cleveland had a hit and freshman Lauren Keller had two RBIs.
In the second game, Keller made her debut in the circle, going seven innings, allowing three hits and striking out eight. At the plate, she had a triple and two RBIs. Reckner hit another home run and had two more hits while Vernae Hillard also had three hits and two RBIs. Katie Hewitt had three hits as well as the Aggie attack had 18 for the game.
Jonesville at Sand Creek: The Aggies started the season off with a 14-2 win over the Comets.
Griffin Reynard went 3-for-3 with a double and scored four runs for the Aggies. Matt Kaufman and Brenden Pickles each went 2-for-2 at the plate and Hunter Wiedyk added a double and had three RBIs.
Noah Benge was the winning pitcher, giving up two runs on six hits in three and a third of an inning. Ryan Lakatos didn't allow a run and struck out three in pitching the other inning and two-thirds.


Starting version 5.5, you can now add virtual column, AKA calculated column, to your existing datagrid. Virtual, by definition, is that it doesn't exist in the database table. It's a calculated field created from other columns. The virtual columns are added to the END of the existing datagrid.
phpGrid only adds a "virtual" column and does NOT change the database table structure. Front-end user should not be able to add/change a column in your database table structure. It should be done only by a very smaller number of people such as DBA, and ultimately through a back-end database administration program.
It's important that virtual column name is NOT an existing database column name used by PHP datagrid.
// n1 stores column 1 value, n2 stores column 7 value..and so on.


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Shop Talk S01E05: What Orchestras Administrators Really Need
Friday, January 1st, 2021 Tuesday, October 13th, 2020 by Drew McManus
23Share on Twitter
One of the best parts of recording episodes in the evening is guests (and host) can actually imbibe. No, this doesn't mean we're having and Drunk episodes, but there's no denying it helps contribute to a discussion sounding more like a group of professionals talking shop.
This ended becoming a fairly broad topic, which I think is best outlined by some of the more memorable quotes:
We all solve problems differently and I don't know if we're always right.
Going into the pandemic, we said that with all these creative minds on staff, if we can't figure this out, nobody can.
At the end of the day, relevance and innovation have to be focal to us.
We have a slew of funders who want to be traditional and don't want to find a new way of doing things so we're beholden to them…and as long as we're shackled to these notions, the industry can't evolve.
Jeff Vom Saal
Jeff is the Executive Director of the Spokane Symphony & Martin Woldson Theater at The Fox, a position he has held since 2016. He is credited with infusing the organization with renewed verve, and has increased revenues from $4MM to over $6MM, an increase of over 50% in three years. He has also spearheaded efforts to broaden the reach of the Symphony and Fox, expanding the range of concerts and events, and establishing a program for children to attend symphony performances for free.
Zak Vassar
Zak is the President & CEO of the Toledo Alliance for the Performing Arts, which consists of the Toledo Symphony and Toledo Ballet. Prior to his work in the arts, he was a marketing consultant, primarily for Fortune 100 firms.
About Shop Talk
The official podcast of Adaptistration.com, Shop Talk invites captivating guests to talk about engaging topics connected to the orchestra business.
Shop Talk Archives | Shop Talk; Last Call Archives
Publication Schedule (subject to change #obvs)
E01: Reaching Diverse Audiences Through The Marcom Lens, Ann Marie Sorrell and Ceci Dadisman 08/18/2020
E02: Art Has Always Been Political, Weston Sprott and Jason Haaheim 09/01/2020
E03: Deconstructing Silos, Anwar Nasir and Scott Harrison 09/15/2020
E04: Fostering BIPOC And Women Composers, Anne M. Guzzo, Daniel Hege, and Holly Mulcahy 09/29/2020
E05: What Orchestras Administrators Really Need, Zak Vassar and Jeff Vom Saal 10/13/2020
E06: The Need For Expertise, Mark Almond and Jason Haaheim 10/27/2020
E07: Changing Your Narrative, Mark Larson and Scott Silberstein 11/10/2020
E08: Centering Equity, Ruby Lopez Harper and Brea M. Heidelberg 11/17/2020
E09: How to Create High-Quality Video Content, Bruce Kiesling and Niccolo Go 12/08/2020
E10: Walking Back Artistic Elitism, Kenji Bunch and Jenny Bilfield 12/22/2020
E11: Being A Content Creator Before It Was Cool, Jeff Curnow 01/05/2021
E12: The Commercial Life, Ceci Dadisman and Marc van Bree 01/19/2021
E13: Composers In Academia, David MacDonald and Sam Merciers 2/2/2021
Shop Talk: Pilot Episode, Poke It With A Stick
Surprise! Today marks the inaugural episode of a new video podcast series where I sit down with movers and shakers in the field and…
Shop Talk Is (Finally) Coming Back
Only 16 months after the first (and only) episode, the Shop Talk video podcast series is finally coming back. Compared to the time between…
The Latest Data Breach
You may have received a data breach notice from an arts organization that uses Blackbaud products. Long story short, Blackbaud was the target of…
Categories Shop Talk Tags Jeff Vom Saal, Zak Vassar Post navigation
An Exciting And Busy Week
Shop Talk Last Call: Cross Pollination In A Good Thing


Dublin, 1914. As Ireland stands on the brink of political crisis, Europe plunges headlong into war.
Among the thousands of Irishmen who volunteer to fight for the British Army is Stephen Ryan, a gifted young maths scholar whose working class background has marked him out as a misfit among his wealthy fellow students. Sent to fight in Turkey, he looks forward to the great adventure, unaware of the growing unrest back home in Ireland. His romantic notions of war are soon shattered and he is forced to wonder where his loyalties lie, on his return to a Dublin poised for rebellion in 1916 and a brother fighting for the rebels. Everything has changed utterly, and in a world gone mad his only hope is his growing friendship with the brilliant and enigmatic Lillian Bryce. The Soldier's Song is a poignant and deeply moving novel, a tribute to the durability of the human soul.
Battered and broken by three years of fighting, Stephen Ryan returns to Ireland – to the woman he loves, and in the hope of a return to his old life.
But, instead, he finds the seeds of a new conflict are being sown in Dublin. Sinn Fein is resurgent, and more determined than ever to gain independence for Ireland. Stephen's own brother is among those who are prepared to fight for their cause, and there is growing civil unrest at the shocking losses of the First World War and the threat of conscription looming over Ireland. With the mood of the whole country changing, Stephen must ask himself if he has chosen the right side. All he knows is that he cannot stay at home. Despite his wounds, and his growing addiction to the morphine he needs to ease his pain, Stephen feels compelled to return to the front, where he has some hope of laying his ghosts to rest and where at least he knows where his loyalties lie. But war is deceitful – whether at home or abroad – and Stephen eventually finds himself dragged into a complex web of deceit and violence. He must think fast, as everything that he holds dear is threatened – this new Ireland has new, unpredictable rules.
London where negotiations are beginning. He leaves behind his brother, Joe, who has been jailed for his actions in the IRA. There are those on both sides who would see the Treaty fail and Stephen soon finds himself beset by problems – a legal dispute, a blackmail attempt, even a plot to assassinate Winston Churchill. This is a story about two brothers, played out against the political and military upheavals that racked Ireland in the 1920s. The Anglo–Irish Treaty brings the war with the British to a close, but a new war is emerging and Stephen finds himself once more called upon as a soldier. Assassinations and guerrilla warfare are the backdrop to the call to arms, as both sides attempt to force a new order.


Each year, carbon monoxide (CO) poisoning is responsible for more than 50,000 emergency department visits1 resulting in more than 400 deaths according to the Centers for Disease Control and Prevention. Americans ages 65 and older are among the highest fatalities. In Utah, there were 224 emergency department visits and two deaths reported in 2017 for CO poisoning. As winter approaches, the risk for CO poisoning increases. The agencies noted in this press release are collaborating to remind Utahns about the symptoms of CO poisoning and provide tips to prevent it.
CO is an odorless, invisible gas produced when gasoline, natural gas, propane, kerosene and other fuels are not completely burned during use. Automobile exhaust is the most common source of CO, but small gas engines, camp lanterns and stoves, charcoal grills, gas ranges, and furnaces also produce it. When appliances and furnaces are improperly adjusted and used in poorly ventilated areas, dangerous amounts of CO can build up in the blood, replacing oxygen and may cause asphyxiation.
Although everyone is susceptible to CO poisoning, unborn babies, infants, the elderly and people with respiratory problems are particularly at risk. Because CO is invisible and odorless, it is important to know the symptoms of CO poisoning and to immediately seek medical help if those symptoms occur.
Have your heating system, water heater, and any other gas, oil, wood, or coal burning appliances serviced by a licensed technician every year. Install an Underwriters Laboratory-approved CO monitor on each level of your home. Check or replace the battery when you change the time on your clocks each spring and fall. Installing a CO monitor should never be a substitute for a professional inspection of home heating and cooking equipment. Owners of boats and recreational vehicles with propane stoves or heaters should install CO detectors.
Do not run a car or truck inside a garage attached to your house, even if you leave the door open.Do not burn anything in a stove or fireplace that is not vented.
* For additional information regarding exposures, hospitalizations and emergency department visits, and deaths relating to CO poisoning in Utah, visit the Utah Environmental Public Health Tracking website at: http://epht.health.utah.gov/epht-view/topic/CarbonMonoxide.html for additional information.


After years of scraping along at virtually zero, U.S. interest rates are starting to shoot higher!
Another force is rising inflation. That's the kiss of death for bondholders because it erodes the value of the fixed payments you get from Treasuries. Still another factor more recently is concern that major foreign debtholders like China will stop buying new U.S. bonds – or heaven forbid, start dumping the massive bondholdings they already have.
Regardless of the CAUSE of the rate increases, the EFFECTS can be found everywhere. Two-year yields just topped 2%, the first time since the 2008 credit crisis that forced the Fed down to zero. The five-year Treasury is at its highest since 2011, above 2.3%, and the 10-year is above 2.5%. Bill Gross, the longtime fund manager nicknamed the "Bond King", is also back in the public eye, declaring on TV that the quarter-century-long bond bull market is dead.
Some are concerned that higher rates will kneecap stocks. But it's too soon for that. Rates are still historically low.
Plus, it's important to remember why the Fed is raising rates: Unlike in 2008 when the economy was on its rear end, it's doing much better now. Unemployment is still very low, hiring is on the mend, and companies just got handed billions of dollars in tax breaks. All that free cash will find its way back into the economy as new hires, raises or other spending, only adding fuel to the fire and making the case for even higher rates.
In other words, stocks of companies with cash on hand, well-managed balance sheets, and solid business models should do fine with higher rates. And banks usually do particularly well in a rising rate environment.
After all, the whole business of banking is marking up the money we deposit by lending it out at higher rates. And if borrowers are confident about the future, they'll borrow and spend despite higher rates.
So to help you find some promising bank investments, I ran a new screen using the Weiss Rating Stock Screener. The first cut, obviously, was to narrow down the list to the banking industry, and sort it so that the highest quality stocks were at the top.
After that, I looked for banks with at least $5 in cash per share, and price-to-earnings-to-growth (PEG) ratios of less than 0.20.
Cash per share is an indicator of how a bank might pay its immediate bills. It counts short-term, liquid investments and cash, and divides that number by shares outstanding. The upshot is that as money gets more expensive, a bank with cash on hand isn't beholden to borrowing to keep the enterprise steady.
The PEG ratio is a way of figuring out what investors will pay for growth. A standard P/E ratio alone shows you that calculation right now; adding in earnings growth gives you a glimpse into the future.
SI Financial Group, Inc. (SIFI, Rated "B+") is the holding company of Savings Institute Bank and Trust Company, a 25-branch lender in Connecticut and Rhode Island. It does mortgage and commercial lending, home equity lending, traditional banking, and wealth management.
The bank stock hasn't done much recently, with a flat performance over the past year. But its three-year return is a much more promising 38.6%. Meanwhile, return on equity over two years has been around 8%, well above the industry average of 2.8%.
The $7.68 per share, in cash on hand and PEG of 0.12 suggest a bank that has been unjustly ignored by investors despite a solid cash position and good prospects for the months ahead, whatever happens with rates.
Bank of Commerce Holdings (BOCH, Rated "A-") is a California retail and commercial bank based in Redding. It has nine offices in the northern part of the state. Like SIFI, this bank seems to have lagged over the past 12 months, returning just 1.8% to investors.
But it has shined over the past five years, with a glowing 172.6% total return. Meanwhile, return on equity is 8.7% and the bank sports a profit margin of 21.8%. It has plenty of cash on hand to weather rate changes, north of $5 per share, and a PEG ratio of 0.19 points to growth at a very reasonable price for investors today.
Bottom line: With interest rates really starting to perk up, bank stocks look even more attractive. So be sure you use our Weiss Ratings Stock Screener to search for good places to invest in the sector. It's a valuable benefit available to our Weiss Ratings Platinum subscribers.
Gavin has more than 30 years of international experience in credit-risk management, commercial lending and insurance, banking and stock analysis and holds an MBA. Gavin oversees the Weiss ratings process, developing the methodology for Weiss' Sovereign Debt and Global Bank Ratings. Gavin has appeared on both radio and television, including ABC and NBC as an expert in insurance, bank and stock ratings and has been quoted by CNBC, The New York Times, Los Angeles Times, and Reuters as well as several regional newspapers and trade media.
© 2019 Weiss Ratings, LLC. All rights reserved.


Over the last couple of years, I've produced a few of the TechNet How Do I? videos for Microsoft and have linked to most (if not all) of them from this blog as they've gone live.
A few days ago, I was reviewing my community activities for the year and noticed one that had slipped through the net: running Hyper-V Server from a USB drive.
Unfortunately it doesn't look as though I'll be doing any more of these as the company I did the work through has lost the contract (and Microsoft produces a lot of this sort of content in house, reducing the scope for outsiders like me). It was a nice gig, while it lasted – if a little time-consuming… hopefully the videos are useful to people!
I like the idea of running Hyper-V from a USB drive for lab / demo purposes, thanks for sharing this.
@Alex – you're very welcome!


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Home Information Application Jura Margulis Imprint
International Piano Academy Freiburg 2017
7/17 – 7/29 2017
Special Project IPAF 2017:
The methodical study, development, and application of an
innovative mnemonic technique for Musical Performance Memory.
Key Elements of the IPAF17:
Individual daily piano master classes with Prof. Jura Margulis in an encouraging atmosphere.
Every participant will receive 4 lessons and access to all events, lectures, publications, excursions, and tickets to the PianoFest.
Languages: English, German, Russian
Lessons with guest artists: Bernd Glemser , Evgeni Koroliov
The unforgettable ambiance of the Historic Kaufhaus concert hall in a 15th century building on Freiburg's medieval Central Square.
A seminar examining the systematic development of virtuoso piano technique.
Lecture: History and Fundamentals of pedagogical traditions from Partimenti to Clementi.
A survey of pianistic traditions from Beethoven through Czerny to Liszt.
Distribution of a compendium of etudes and exercises drawn from rare historical material made exclusively for participants of the IPAF17.
Individual lessons in which various methods are applied to the participants' repertoire.
A seminar examining historical keyboard instruments.
Excursion to the Fritz Neumeyer Collection of Historical Keyboard Instruments at the Schloss Bad Krozingen and presentation of more than 30 playable keyboard instruments from the 15th to the 19th centuries.
Demonstration of unique Welte-Mignon recordings on one of the last functional piano-roll instruments.
The Welte-Mignon company was founded in 1872 and attracted such artists as Claude Debussy, Camille Saint-Saëns, Alexander Scriabin, Max Reger, Edvard Grieg, George Gershwin, Ignacy Paderewski, and Ferruccio Busoni to make piano-roll recordings in Freiburg, the company's headquarters.
Info-workshop with an associate of a major concert agency.
Lecture and individual consultation with Mr. Andreas Vierziger, an artist manager formerly working at Mark Stephan Buhl Artists Management, Vienna.
Numerous public performance opportunities for all participants throughout the IPAF16.
Active participants have the opportunity to perform in front of a wide audience in public recitals in and around Freiburg, including the City Hall of Bad Krozingen, the Dommusikschule, and the Kaisersaal of the Historic Kaufhaus. The selection of performers and repertoire is in the discretion of the IPAF organisation.
The PianoFest, a prestigious concert series.
Since 1986 a recital series has taken place in parallel with the master classes. Over the years, many artists including Martha Argerich, Paul Badura-Skoda, Dmitry Bashkirov, Elizabeth Leonskaja, Vitaly Margulis, Bella Davidovich, Malcolm Bilson, Lylia Zilberstein, Peter Donohoe, Bernd Glemser, Igor Kamenz, Sergey Babayan, Boris Bloch, Kirill Gerstein, Ingrid Fliter, Olli Mustonen, Polina Leschenko, and Awadagin Pratt have performed in this series. The final concert of the participants is also the final concert of the PianoFest concert series.
MON 7/17, 8:15 PM: Opening Recital, Jura Margulis
SAT 7/22, 8:15 PM: Piano Recital, Bernd Glemser
THU 7/27, 8:15 PM: Piano Recital, Evgeni Koroliov
SAT 7/29, 7:00 PM: Final Recital of the Participants of the IPAF17
Download the PianoFest Programm 2017 as a PDF (June)
An audience-vote competition at the final participants' recital:
At the end of the master class, participants perform in a recital at which the audience votes for their favorites to receive cash prizes.
Laureates of the audience-vote competition of the IPAF 2016:
1. prize:
Program subject to change
For more detailed information concerning the IPAF16 please contact info@internationalpianoacademy.org
Article about the International Piano Academy Freiburg 2009 in PianoNews
Article about the pedagogical work with Rex Lewis IPAF 2011 in Cultur.Zeit
TV Südbaden/YouTube: Genialer Autist am Klavier
▲ Historisches Kaufhaus ▲
▲ Freiburg Cathedral ▲
▲ Historic Instruments▲
▲ PianoFest 2017 ▲
▲ Paul Badura-Skoda 2009▲
▲ Student Recital ▲


text:
the strange Archives
Outland Entertainment Brings Matt Forbeck's Shotguns & Sorcery Novels To Tabletop RPGs
The game will use the Cypher System from Monte Cook Games, as featured in The Strange RPG and the multiple-award winning Numenera (also the basis of the video game Torment: Tides of Numenera coming from inXile). The Shotguns & Sorcery RPG will be designed by Robert J Schwalb, who was recently a designer on the fifth[...]
Want To Play A Game Run By Monte Cook Or Bruce Cordell At Gen Con This Year?
By Christopher Helton Monte Cook Games is going to have a charity auction, giving away chances to play in a Numenera game run by Monte Cook or a game of The Strange run by Bruce Cordell at Gen Con 2014. [Monte Cook] Numenera is a science fantasy roleplaying game, set in a far future setting known as The Ninth World[...]
Where Else Will We Find The Strange Woman Of The New 52?
So Bleeding Cool has been noting and looking out for appearances of The Strange Woman who first appeared in Flashpoint #5, through the new DC relaunch titles But does her influence go further back? Weve been looking through the our back issues… Superman #75 was a big moment for DC and one of their biggest icons[...]
All Thirteen Appearances Of The Strange Woman In This Week's DC Comics
Here are all thirteen appearances of The Strange Woman Of Flashpoint, appearing in all the DC Universe titles shipping this week. From Batgirl #1. From Swamp Thing #1. From O.M.A.C #1 From Green Arrow #1. From Static Shock #1. From Detective Comics #1. From Stormwatch #1. From Men Of War #1. From Batwing #1. From Action Comics[...]
The Spawnies Awards Releases List Of 2023 Winners
AEW Fight Forever Delayed Due To ESRB Rating Issues


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The shoot was scheduled for Thursday Jul 21, 2016. Location; Aqua Safari, Ada, we left home at 6am too meet up with the rest of the team from the designer's side (Totally Ethnik) at the Accra Mall.
4 hours later, we arrived at our destination. The rest of the team joined us 1 hour later. My Assistant (Kwame Prosper aka The Blue Dee) and I scouted the place for possible places to shoot at. Aqua Safari provided us with many options, ranging from a beach scene to classic wooden and brick backgrounds as well as lots of greenery, not forgetting a swimming pool and boat themed buildings.


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7 most dangerous toxins in vaccines of at least 60 toxins • Jetsun Sunbeds Advice • Tanning Salon Burnley, Lancashire
7 most dangerous toxins in vaccines of at least 60 toxins
by Admin » 08 Mar 17 20:16
The 7 most dangerous vaccines injected into humans and exactly why they cause more harm than good
Wednesday, December 14, 2016 by: S.D. Wells
flu shot, mercury, MMR, polio, rotavirus, vaccine damage, vaccine toxins
(Natural News) Oh, the theory of vaccines sounds great. Inject a tiny bit of the live virus into your blood so you can build antibodies and thus immunity against the "real deal" later. If that's all there was to it, it could actually work. Then there's the fear mongering that's thoroughly "inflamed" and propagated by the press, pharma, and the medical doctors of quack Western medicine. This is where the real money is made. If you get measles you could die! If you get polio you'll surely be paralyzed for life! If you get Zika, your baby's head will be shrunken and deformed!
Yet, what if you found out today that the worst odds you or your children have of being infected with disease, disorder, and deformity exist in getting injected repeatedly with neurotoxins, genetically modified bacteria, live experimental strains of multiple viruses and pesticides? Consider this:
not one single vaccine ever produced that is recommended by the CDC today has ever been proven safe or effective. Why? They don't have to prove it. All they have to do is scare the living hell out of everyone using propaganda, and it's worked for 75 years.
Presenting the 7 most dangerous vaccines injected into humans without any proof of safety or efficacy
#1. Gardasil HPV – Forget for a moment the fact that many girls who get the HPV vaccine beginning at age 9 for a sexually transmitted disease (diseases they don't have) go into immediate anaphylactic shock and some into comas and die, and let's just talk about the insane boatload of chemicals the manufacturers put in this concoction that belong nowhere in medicine, ever, especially that which is injected directly into muscle tissue and that which can penetrate the blood/brain barrier. Plus, remember to triple the amounts of these carcinogenic, dangerous, ludicrous chemical ingredients of Gardasil, because there are 3 of these toxic jabs required.
First we have sodium borate at 35mcg. Also known as "borax," this is the main poisonous ingredient in boric acid that's used to kill cockroaches. Is your little girl a cockroach? Is it coincidence that the side effects listed and reported with the Gardasil vaccine match those of sodium borate poisoning? No, it's not a coincidence. Did you know that anything imported into the European Union that contains borax must carry a warning label stating, "May damage fertility" and "May damage the unborn child." This is what America "recommends" for preteen and teenage girls who are just reaching the age of fertility. Unbelievable!
Then, Gardasil HPV contains aluminum at 225mcg, which causes nerve cell death and helps the vaccine chemicals enter the brain. Let's not forget that Gardasil HPV contains polysorbate 80 at 50mcg. Polysorbate 80 is used as an emulsifier in foods, but when injected into animals (such as humans), causes rapid, unnatural growth of reproductive organs, causing sterility. This is population control through vaccines, just as Bill Gates once said at a TED conference would be ideal for reducing the world's population by a few billion. Polysorbate 80 is what causes the anaphylactic shock and also causes cancer and birth defects, while we're on that topic. Sorry, but there's not enough time to talk about the sodium chloride at nearly 10mcg.
#2. Anthrax vaccine (biothrax) – The dreaded anthrax jab contains aluminum hydroxide, formaldehyde (yes, embalming fluid for the dead), and benzethonium chloride. In 2009, a study published in the Journal of Inorganic Biochemistry stated that aluminum hydroxide could be the primary cause of Gulf War Syndrome. Aluminum hydroxide causes apoptosis of motor neurons, leading to dementia. Go figure. Thousands of US soldiers given the mandatory anthrax jab are still sick or have died. It was never approved by the FDA, yet any soldier refusing it got dishonorably discharged, fines, and possible prison time. President Clinton's executive order 13139 gave the DoD permission to experiment on the US military with the highly dangerous anthrax concoction.
#3. MMR II – Under Appendix B, listed on the CDC website, you can find the ingredients for the MMR (MMR-II), the combination vaccines that contain recombinant human albumin, sorbitol, hydrolized gelatin, chick (egg) embryo cell culture, human diploid lung fibroblasts, and fetal bovine serum, among other certain preservatives and chemical adjuvants. In the "ProQuad" version, or MMRV (w/vericella for chicken pox), they've added monosodium L-glutamate, neomycin, and MRC-5 cells. And although measles is a respiratory disease accompanied by an uncomfortable rash and fever illness that anyone with a normal immune system will likely survive, the media scares the public into getting jabbed with neurotoxins.
Sorbitol is a synthetic sweetener which metabolizes very slowly and aggravates IBS and gastrointestinal issues. Fetal bovine cow serum is extracted from cow skin and when injected causes connective tissue disorders, arthritis and lupus; also shortness of breath, low blood pressure, chest pain and skin reactions. Sodium chloride raises blood pressure and inhibits muscle contraction and growth. Human albumin is the protein portion of blood from pooled human venous plasma and when injected causes fever, chills, hives, rash, headache, nausea, breathing difficulty, and rapid heart rate. Injecting "pooled blood" can result in a loss of body cell mass and cause immunodeficiency virus infection, or contain SV40, AIDS, cancer or Hepatitis B from drug addicts. Still want that MMR vaccine? Didn't think so.
#4. Swine Flu – This loaded nightmare hoax vaccine contains inactivated H1N1 virus propagated in embryonated chicken eggs. The multi-dose vials contain over 24mcg of mercury per .5 ml dose! The jab also contains antibiotics polymyxin and neomycin that annihilate good gut bacteria, making the immune system highly vulnerable to infection. Add in some fluid from chicken eggs and you have one of the most experimental jabs ever created and a hoax perpetuated by WHO, GSK and the CDC to profit in the billions.
#5. Polio – This psycho-jab contains inactivated monkey kidney cells, newborn calf serum, embalming fluid, antibiotics, and bovine albumin. Salk didn't invent the cure for polio–he invented new strains of it by haphazardly combining several. Get the facts!
#6. Influenza vaccine (a.k.a. the flu shot) – Specifically, the "FluLaval" flu shot contains 25 mcg of mercury in one jab. The EPA safety limit for drinking water? Just 5 mcg. Do the maths, then consider that shots bypass digestion, breathing, and skin filters. Common flu jabs also contain formaldehyde and polysorbate 80.
#7. RotaTeq for Rotavirus – Three oral doses of this Merck-made horror story cost about $200 and are mandated for about four million infants every year. Rotavirus vaccine contains 5 live strains, plus some fetal bovine serum and porcine circovirus–a volatile and dangerous virus that infects pigs. Side effects of RotaTeq? Difficulty breathing, vomiting and ear infection, followed by bloody stool. Then the intestines get blocked and twisted (known as intussusception) which can be deadly and requires surgery on infant's intestines. Be sure and call your doctor right away if your child dies from RotaTeq.
7 most dangerous toxins in vaccines of at least 60 toxins • Tanning and Health News