text:
Next in The JI
Pillars of Immunology
Translating Immunology
Annual Meeting Abstracts
COVID-19/SARS/MERS Articles
Influence Statement
ImmunoCasts
AAI Disclaimer
American Association of Immunologists
ImmunoHorizons
Follow The Journal of Immunology on Twitter
Follow The Journal of Immunology on RSS
Anergic T Cells Are Metabolically Anergic
Yan Zheng, Greg M. Delgoffe, Christian F. Meyer, Waipan Chan and Jonathan D. Powell
J Immunol November 15, 2009, 183 (10) 6095-6101; DOI: https://doi.org/10.4049/jimmunol.0803510
Greg M. Delgoffe
Christian F. Meyer
Waipan Chan
Jonathan D. Powell
Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.
The response of a T cell to a particular Ag is determined by the context in which the Ag is encountered (1). In the two-signal model, TCR engagement (signal 1) results in full T cell activation only when recognition occurs in the setting of costimulation (signal 2). Signal 2 is typically delivered when B7 molecules on the surface of activated APCs engage CD28 on the surface of the T cell. In the absence of costimulation, Ag recognition fails to promote full cytokine release and proliferation. Furthermore, the T cells enter a state of hyporesponsiveness known as anergy. Anergic T cells fail to elaborate cytokines and proliferate even when stimulated under normally activating conditions (signals 1 plus 2) (2).
Upon activation, the bioenergetic demands of a T cell increase dramatically over the resting state. Activated T cells are highly anabolic and demonstrate a marked increase in glycolysis (3). T cells will employ glycolysis to generate ATP even in the presence of high levels of oxygen. With activation there is an increase in glucose and amino acid uptake and suppression of catabolism. While there has been much focus on the role of costimulation in promoting T cell effector function, only recently has its role in regulating metabolism become apparent. CD28-induced activation of Akt increases glucose uptake by promoting the translocation of the glucose transporter glut1 to the cell surface (4). CD28 stimulation also promotes the activation of mammalian target of rapamycin (mTOR)4 (5). mTOR, through its downstream substrates S6K1 and 4E-BP1, promotes translation and protein synthesis (6). Likewise, mTOR facilitates increased amino acid uptake by increasing the cell surface expression of amino acid transporters. T cell activation requires increased energy utilization, and, as such, a role for the energy sensor AMP kinase in regulating T cell function is becoming apparent (7). AMP kinase is activated under conditions of stress and leads to the inhibition of energy consuming processes. AICAR, an activator of AMP kinase, has been shown to suppress T cell activation (8).
Our laboratory is interested in understanding the signaling pathways that regulate T cell activation vs tolerance (9). During the course of our studies we have identified mTOR as playing a critical role in integrating costimulatory signals (5, 10). Our group and others have demonstrated that for Th1 T cells, inhibition of mTOR promotes T cell anergy, even in the presence of signals 1 plus 2 (11, 12). Since mTOR is a central regulator of protein synthesis and metabolism, we wanted to investigate the status of metabolic pathways in anergic T cells. Our data indicate that not only are anergic Th1 T cells unable to up-regulate metabolic machinery in response to signals 1 plus 2, but inhibition of metabolism can lead to T cell anergy.
Reagents and Abs
N-acetyl-leucine amide (NALA) was purchased from Bachem and dissolved in ethanol. 2-Deoxyglucose (2DG) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) were obtained from Sigma-Aldrich and dissolved in PBS. Rapamycin was purchased from Sigma-Aldrich and dissolved in ethanol. Phospho-S6K1 (Thr421/Ser424), phospho-Akt (S473), and total S6K1 were obtained from Cell Signaling Technology. Total Akt was obtained from Santa Cruz Biotechnology. Anti-rabbit IgG HRP-linked Ab was obtained from Amersham Biosciences.
Cell culture and mice
A.E7, a CD4+ Th1 clone specific for pigeon cytochrome c (PCC) peptide 81–104 (FAGIKKKAERADLIAYLKQATAK), was maintained as previously described (11). Briefly, A.E7 cells were maintained in 50% RPMI 1640 and 50% EHAA supplemented with 10% FBS, 2 mM glutamine, 50 μM 2-ME, 100 IU/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 50 μg/ml gentamicin (Invitrogen). To expand these cells, they were stimulated for 48 h with 5 μM PCC peptide (Sigma-Aldrich) and irradiated (3000 rad) B10.A mouse splenocytes (with a ratio of A.E7 cell/B10.A splenocyte as 1:10). Mouse rIL-2 (1 ng/ml; Invitrogen) was then added to the culture. After 10 days, live cells were isolated by a Ficoll gradient (Sigma-Aldrich). B10.A mice were purchased from Taconic. 5C.C7 mice, RAG2−/− mice with a transgenic TCR specific for PCC peptide 81–104, were obtained from Taconic. To generate previously activated 5C.C7 T cells, splenocytes from these mice were incubated with 5 μM peptide for 48 h and rested in IL-2 for 5 days. Live T cells were isolated by a Ficoll gradient. The studies have been reviewed and approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.
For all analyses, cells were stained in 1× PBS, 2% FBS, 0.02% sodium azide solution. Abs to CD4, CD98, and CD71 were purchased from BD Biosciences. Live cells were stained for 15 min at 4°C and fixed in 4% PFA. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest software.
Cytokine and lactate detection
Cytokines were measured from cell culture supernatants using the Ready-SET-Go! murine IFN-γ and IL-2 kits from eBioscience. Lactate was measured from supernatants using the EnzyChrom l-lactate assay kit from BioAssay Systems.
Anergy model
To induce anergy, T cells were treated with plate-bound anti-CD3 (3 μg/ml, 2C11; BD Biosciences). To activate, T cells were stimulated with plate-bound anti-CD3 plus soluble anti-CD28 (2 μg/ml, 37.51; eBioscience). After 16 h, the cells were washed three times with PBS to remove Abs and rested in media for 5–7 days. Upon rechallenge, 5 × 104 T cells were stimulated with 5 × 105 irradiated B10.A splenocytes and PCC peptide (0–0.5 μM), and proliferation was measured by thymidine incorporation after 48 h. To determine IL-2 production upon rechallenge, 4 × 105 treated cells were stimulated with plate-bound anti-CD3 (1 μg/ml) plus soluble anti-CD28 (2 μg/ml), supernatants were collected after 24 h, and IL-2 was measured by ELISA (eBioscience).
Immunoblot analysis
T cells were harvested and washed with ice-cold PBS. The cells were lysed with whole cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 1× protease inhibitor cocktail (Roche). After 20 min of incubation on ice, cell lysates were spun at 15,000 × g for 5 min, and supernatant was collected. Protein concentration was assessed using a BCA protein assay kit (Pierce). Next, equal amount of cell lysates (20–40 μg) were loaded onto NuPAGE minigels (Invitrogen), separated by SDS-PAGE, and transfered to nitrocellulose or polyvinylidene difluoride membrane (Invitrogen). The membrane was then blocked with TBS with 5% nonfat dry milk and 0.1% Tween 20 and probed with indicated Abs according to the manufacturer's instructions.
The induction of anergy is associated with incomplete up-regulation of metabolic machinery
When the CD4+, Th1, PCC-specific T cell clone A.E7 is stimulated with anti-CD3 in the absence of costimulation, such cells demonstrate a marked decrease in their proliferative capacity and ability to produce IL-2 (Fig. 1⇓, A and B). From a metabolic perspective, A.E7 T cells stimulated with signal 1 alone demonstrate decreased up-regulation of the amino acid transporter CD98 (Fig. 1⇓C). Likewise, they do not maximally up-regulate the transferrin receptor (CD71), which is responsible for the uptake of iron (Fig. 1⇓D). mTOR activation, which is critical for cellular metabolism, proceeds by two downstream signaling complexes: TORC1, which is associated with the adaptor protein raptor, and TORC2, which is associated with the adaptor protein rictor (13). Downstream TORC1 signaling leads to the phosphorylation of S6K1, while TORC2 activation leads to phosphorylation of Akt at S473. Stimulation with anti-CD3 alone fails to fully up-regulate TORC1 activity as determined by the phosphorylation of S6K1 (Fig. 1⇓E). Likewise, as has been previously shown, Akt activity (14), which plays a role in glucose metabolism in T cells, fails to become fully up-regulated by signal 1 alone (Fig. 1⇓F). Thus, when stimulated with signal 1 alone (anti-CD3), A.E7 T cell clones not only fail to be fully activated but also demonstrate defects in the metabolic machinery necessary for glucose, amino acid, and iron metabolism.
Costimulation is required for full T cell activation and up-regulation of metabolic machinery. A, A.E7 T cells were left unstimulated or treated with anti-CD3 alone (signal 1) or anti-CD3 plus anti-CD28 (signals 1 plus 2) for 16 h, then interrogated for proliferation by thymidine incorporation. B, Supernatants from A were interrogated for IL-2 by ELISA. C, CD98 cell surface expression of cells treated as in A by FACS. The shaded tracing indicates the isotype control for stimulated T cells. D, As in C, but for CD71. E, Resting A.E7 T cells were left unstimulated or stimulated through the TCR (anti-CD3) or TCR and costimulation (anti-CD3 plus anti-CD28) for 3 h and interrogated for phospho-S6K1. F, As in E, but for phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. Data are representative of three independent experiments.
Anergic T cells fail to up-regulate metabolic machinery upon rechallenge
When A.E7 cells are stimulated with anti-CD3, rested, and then rechallenged with full stimulation, such cells are hyporesponsive; they are anergic. As we and others have previously shown, full stimulation of anergic Th1 T cells act functionally as if they were only stimulated with anti-CD3 alone. Since anti-CD3 alone is insufficient to support T cell metabolism, we next examined the regulation of metabolic machinery in the anergic cells. A.E7 T cells were left unstimulated or rendered anergic with anti-CD3 stimulation alone, rested in fresh media for 7 days, and rechallenged. Upon rechallenge such cells fail to produce IL-2 and proliferate (Fig. 2⇓, A and B). Next, we sought to assess the metabolic potential of anergic verus nonanergic cells. Since it has been shown that the glycolytic pathway plays a critical role in providing energy for activated T cells, we interrogated anergic and nonanergic T cells for their ability to produce lactate upon rechallenge (14). T cells that had been given initially full stimulation produce a basal level of lactate while resting (first bar of Fig. 2⇓C). Upon activation with signals 1 plus 2, anergic T cells (cells that had previously been activated with signal 1 alone) produce slightly higher levels of lactate when compared with the unstimulated cells (Fig. 2⇓C). On the other hand, T cells that had been activated with signals 1 plus 2 (nonanergic T cells) and then rechallenged 7 days later with signals 1 plus 2 produced more lactate than did both the unstimulated cells (resting cells vs activated, p < 0.01) and the rechallenged anergic cells (anergic vs activated, p < 0.001). It has previously been demonstrated that culturing anergic T cells in IL-2 reverses anergy (15). Thus, we wanted to determine whether IL-2 also reversed the metabolic defects seen in anergic cells. Indeed, cells that had been previously rendered anergic with signal 1 alone and then cultured in IL-2 demonstrated high levels of lactate production (anergic vs anergic plus IL-2, p < 0.01; anergic plus IL-2 vs activated, p > 0.10), consistent with the fact that IL-2 reverses anergy.
Upon rechallenge with signals 1 plus 2, anergic T cells fail to not only produce IL-2 and proliferate but up-regulate metabolic machinery. A, Mock-treated and anergic (signal 1) A.E7 T cells were rechallenged with APCs plus PCC peptide for 48 h. Cells were then interrogated for proliferation by thymidine uptake. B, Mock-treated and anergic (signal 1) T cells were rechallenged with anti-CD3 and anti-CD28 for 16 h and interrogated for IL-2 by ELISA. C, Supernatants from rechallenged T cells were tested for l-lactate production. For these experiments some cells were left unstimulated, and some anergic T cells were rescued with high-dose IL-2. D, CD98 cell surface expression of cells treated as in B by FACS. The shaded tracing indicates the isotype control for the stimulated T cells that were initially mock treated. E, As in D, but for CD71. F, As in D and E, but some anergic cells were rescued with IL-2, G, Mock-treated or anergic A.E7 T cells were left unstimulated or were stimulated for 3 h with anti-CD3 and anti-CD28; lysates were made and probed for phospho-S6K1 by immunoblot. H, As in G, but for phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. Data are representative of three independent experiments. Statistics were performed using the Student's t test. ∗∗, p < 0.01; ∗∗∗, p < 0.001; NS, p > 0.05.
In as much as the anergic cells failed to mount a strong glycolytic response, we wanted to examine their ability to up-regulate other aspects of metabolism upon rechallenge. As such, anergic and nonanergic ("mock" treated during induction) T cells were assayed for their ability to up-regulate the amino acid transporter CD98 and the transferrin receptor CD71 (Fig. 2⇑, D and E). Previously, we have demonstrated that clonotypic T cells with low CD71 expression could be used as a crude way to identify anergic T cells in vivo (5). In both cases, in the resting state, these receptors were equivalently expressed between the nonanergic and anergic T cells. When the cells were then rechallenged with full stimulation (anti-CD3 plus anti-CD28), the nonanergic (mock) T cells up-regulated both CD98 and CD71 (Fig. 2⇑, D and E). However, when the anergic T cells were rechallenged with full stimulation (anti-CD3 plus anti-CD28) they failed to fully up-regulate CD98 and CD71 (Fig. 2⇑, D and E). Alternatively, when anergic cells are rescued with high-dose IL-2 (2 ng/ml) during the rest period, these cells regain their metabolic activity and their ability to up-regulate these metabolic receptors (Fig. 2⇑F). Note that the up-regulation of both CD98 and CD71 in the anergic cells rescued in IL-2 overlaps with that of the rechallenged nonanergic (mock-treated) cells. Likewise, anergic T cells failed to fully activate TORC1 as measured by S6K1 phosphorylation and TORC2 as measured by Akt S473 phosphorylation (Fig. 2⇑, G and H). Thus, in addition to being functionally hyporesponsive, anergic T cells are hyporesponsive in terms of their ability to up-regulate metabolic machinery. From a bioenergetic perspective, anergic T cells are metabolically anergic as well.
Inhibition of metabolic pathways mitigates T cell function
Our data demonstrate that when anergic T cells are stimulated with full stimulation (signals 1 plus 2), they fail to completely up-regulate metabolic machinery necessary for amino acid, glucose, and iron metabolism. Our findings raised the possibility that the failure of anergic cells to fully up-regulate metabolic machinery was not just a consequence of anergy but perhaps contributed to the functional anergic phenotype. For example, whereas a hallmark of T cell clonal anergy is the inability to produce IL-2 and IFN-γ upon rechallenge, our data suggested that the block in the production of these two cytokines may be enforced in part by the inability of anergic cells to adequately increase metabolism. Thus, we wanted to determine the effect of limiting amino acids, glucose, and energy on IL-2 and IFN-γ production.
To examine this, we examined the effect of blocking metabolic pathways on T cell activation. We first tested whether signals 1 plus 2 could activate T cells in the presence of amino acid deprivation. The branched-chain amino acid leucine is the most abundant amino acid in dietary proteins. Furthermore, the inability of the liver to catabolize leucine prevents its "first-pass" loss, allowing leucine to act as a nutrient signal (16). Alteration in leucine abundance would have a considerable impact on amino acid-derived signaling.
To elucidate the role of leucine in T cell activation, we stimulated T cells in the presence of the leucine antagonist NALA. NALA mimics leucine depletion by acting as a competitive antagonist (17). Primary 5C.C7 T cells were stimulated for 2 days with peptide and then expanded for an additional 5 days in IL-2 to generate previously activated primary Th1 cells. These cells were rechallenged with anti-CD3 plus anti-CD28 in the presence of increasing doses of NALA. From a technical perspective, stimulating the T cells with Abs enabled us to eliminate a potential affect of leucine antagonism on APCs. By functionally decreasing available leucine with NALA, there is a dose-dependent inhibition of both proliferation (data not shown), IL-2, and IFN-γ production (Fig. 3⇓, A and B). Notably, the decrease in function was not associated with a decrease in viability as measured by trypan blue (data not shown). Thus, in nonanergic cells, limiting amino acid metabolism leads to a decrease in Th1 cytokine production. Intimately linked to the availability of nutrients is energy in the form of ATP. The status of cellular energy stores is sensed by AMP-activated protein kinase (AMPK) (18). Energy insufficiency increases the ratio of AMP/ATP, leading to an interaction between AMP and a regulatory subunit of AMPK. This association induces a conformational change in AMPK that allows AMPK to be phosphorylated and activated by its upstream protein kinases (19). Thus, activation of AMPK signals the cells that their energy supply is insufficient. Here, we employed a well-known activator of AMPK, AICAR, to imitate energy deprivation (18). As with NALA, AICAR inhibited IL-2 and IFN-γ production in a dose-dependent fashion (Fig. 3⇓, A and B). Once again, the decrease in function was not associated with an increase in cell death.
Metabolic inhibition leads to decreased T cell function. Previously activated (Th1) 5C.C7 T cells were stimulated in varying concentrations of 2DG (mM), NALA (mM), or AICAR (μM). Supernatants were then interrogated for (A) IL-2 or (B) IFN-γ. Data are normalized values from three independent experiments. Error bars indicate SD. C, Metabolic inhibition induces anergy even in the presence of costimulation. A.E7 T cells were stimulated 16 h with anti-CD3 alone or anti-CD3 plus anti-CD28 in the presence of rapamycin (200 nM), NALA (2 mM), AICAR (10 μM), or 2DG (500 μM). The cells were washed and rested without drug for 7 days in unsupplemented media or media containing 1 ng/ml murine IL-2. The cells were then rechallenged for 16 h with anti-CD3, anti-CD28, without any drugs, and interrogated for IL-2 production by ELISA. Error bars indicate SD. Data are representative of three independent experiments.
Anergic T cells displayed a decrease in Akt activation, which is crucial for the full up-regulation of glucose metabolism (4). As such, we wanted to determine the effect of inhibiting glucose metabolism on T cell function. 2DG, an antagonist of glucose, is able to compete with glucose for the same transporters on the cell surface, and hexokinase, which catalyzes the first step of glycolysis in the cytoplasm (21). However, the product, 2-deoxy-glucose-6-phosphate, cannot be further processed. Therefore, 2DG inhibits both the uptake and utilization of glucose. When 5C.C7-derived Th1 cells were fully activated (anti-CD3 plus anti-CD28) in the presence of increasing concentrations of 2DG, we observed a decrease in IL-2 and IFN-γ production that was not associated with increased death (Fig. 3⇑, A and B). When compared with leucine and energy depletion, inhibiting glucose metabolism appeared to be a more potent means of inhibiting T cell function. Overall, these data demonstrate that limiting branched-chain amino acid, energy, and glucose in Th1 T cells can inhibit the production of Th1 effector cytokines.
Inhibition of metabolism induces anergy even in the presence of costimulation
T cells stimulated with signal 1 alone fail to proliferate, produce IL-2, and are anergic upon rechallenge. The induction of anergy was associated with inhibition of the metabolic machinery. Likewise, our data in Fig. 3⇑, A and B, demonstrate that inhibition of metabolism leads to inhibition of T cell function. In the next series of experiments we tested if inhibition of the metabolic machinery promoted the induction of anergy. Specifically, we sought to determine whether stimulation of A.E7 T cells in the presence of NALA, 2DG, or AICAR resulted in anergy. To investigate this, A.E7 cells were stimulated with signal 1 alone or signals 1 plus 2 in the absence or presence of a metabolic inhibitor (NALA, 2DG, or AICAR). Sixteen hours later, the cells were washed extensively to remove anti-CD3 and anti-CD28 Abs and the inhibitors, and then rested in fresh unsupplemented media for 7 days. The cells that were initially treated with signal 1 alone were rendered anergic, as they produced less IL-2 upon full rechallenge (Fig. 3⇑C). In contrast, the cells that were stimulated with signals 1 plus 2 during induction remained active upon rechallenge, secreting a large amount of IL-2. The cells that were treated with metabolic inhibitors in the earlier stimulation were hyporesponsive upon rechallenge in the absence of the inhibitors; they were rendered anergic. Thus, metabolic deficiency promoted anergy even in the presence of costimulation.
Interestingly, we observed that anergy could be induced at doses of the metabolic inhibitors (NALA (2 mM), AICAR (10 μM), or 2DG (500 μM)) that had minimal affect on cytokine production (note that at these doses in Fig. 3⇑, A and B, there is minimal inhibition of IL-2 and IFN-γ). However, these low doses of the inhibitors were still able to induce a state of anergy such that upon rechallenge (in the absence of the inhibitors) the cells failed to produce IL-2. That is, even though the inhibitor-treated cells produced copious amounts of IL-2 during the initial stimulation they were still rendered anergic and failed to produce IL-2 upon rechallenge 7 days later (Fig. 3⇑C). Thus, the metabolic inhibitors were able to induce anergy even in the presence of IL-2, suggesting that they were acting downstream of IL-2R signaling.
A hallmark of anergy is its ability to be reversed by addition of IL-2. We sought to test whether T cells rendered anergic by metabolic inhibition could be rescued by IL-2. A.E7 T cells were stimulated with signal 1 alone, signals 1 plus 2, or signals 1 plus 2 with drug as previously described. Cells were washed and rested in either unsupplemented media or media containing 1 ng/ml murine IL-2. Seven days later, the cells were all rechallenged with signals 1 plus 2 and assessed for IL-2 production. While cells receiving signal 1 alone are rendered anergic, those cells that received IL-2 during the rest period were rescued (Fig. 3⇑C). Furthermore, cells rendered anergic by metabolic inhibition were also rescued by addition of IL-2. Thus, although metabolic inhibition can promote the induction of anergy even in the presence of costimulation and IL-2 production, cells rendered anergic in the presence of metabolic inhibitors can be rescued from anergy by being cultured in IL-2 in the absence of the inhibitors.
Previously, we and others have shown that for Th1 cells, TCR engagement in the absence of mTOR activation leads to anergy (Fig. 3⇑C, signals 1 plus 2 plus rapamycin) (11, 12). Thus, we hypothesized that the ability of the metabolic inhibitors to induce anergy might be due to their ability to inhibit mTOR. 5C.C7 T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of rapamycin, NALA, 2DG, and AICAR. Extracts from these cells were interrogated for mTOR activity by immunoblot analysis (Fig. 4⇓A). TORC1 activity results in the phosphorylation of S6K1 and, in addition to rapamycin, each of the inhibitors (at the doses that induce anergy) blocks S6K1 phosphorylation. TORC2 activation results in the phosphorylation of Akt at S473. Each of the inhibitors blocked phosphorylation of Akt at S473, demonstrating their ability to block TORC2. For AICAR, we hypothesized that this inhibition of mTOR was mediated through the activation of AMPK. Unstimulated T cells display essentially no phosphorylation of AMPK (Fig. 4⇓B). Stimulation with signals 1 plus 2 leads to a slight increase in AMPK phosphorylation but a robust increase in mTOR activation as measured by phosphorylation of S6K1. On the other hand, as has been previously described, AICAR markedly enhances AMPK phosphorylation and inhibits mTOR (Fig. 4⇓B) (20). Thus, mechanistically, the ability of these compounds to induce anergy correlates with their ability to inhibit mTOR activation.
Full T cell stimulation in the presence of metabolic inhibition leads to tolerance induction via mTOR inhibition. A, NALA, 2DG, and AICAR mimic starvation signals in an mTOR-dependent fashion. Previously activated 5C.C7 T cells were left unstimulated or stimulated with anti-CD3 plus anti-CD28 (TCR + Costim) in the presence or absence of NALA (2 mM), 2DG (500 μM), AICAR (10 μM), or rapamycin (200 nM) for 3 h, then lysed and interrogated for phospho-S6K1 and phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. B, 5C.C7 T cells were left unstimulated or were stimulated with anti-CD3 and anti-CD28 (TCR + Costim) with or without AICAR (10 μM); lysates were made and probed for phospho-AMPK, phospho-S6K1, and pan S6K1 by immunoblot. Immunoblots are representative of at least three independent experiments.
Our observations demonstrate that the T cell metabolic machinery is regulated in anergy and that regulation of T cell metabolism contributes to decreased effector function of anergic T cells. Phenotypically, anergic T cells fail to maximally express effector cytokines. In this report we demonstrate that the ability to up-regulate metabolic machinery is also inhibited in anergy. Since impaired T cell metabolism prevents full T cell effector function, our data raise the possibility that anergy is in part maintained by mitigating the up-regulation of T cell metabolic machinery. Clearly, our report does not exhaustively examine all of the metabolic changes that occur upon T cell activation. We have shown that glycolysis, as measured by lactate production, up-regulation of the amino acid transport CD98, up-regulation of the transferrin receptor CD71, Akt activation necessary for full glucose metabolism, and mTOR activation necessary for protein translation are all inhibited in anergic cells stimulated with signals 1 plus 2. As such, we propose that the term anergy (the inability to respond upon rechallenge) can be applied to not only a lack of cytokine production and proliferation in Th1 cells but also to a lack of up-regulation of the metabolic machinery. Future investigation will determine the full scope of metabolic anergy.
Additionally, we demonstrate that blocking leucine, glucose, and energy metabolism not only acutely inhibits T cell function but also promotes the induction of T cell anergy in Th1 T cells. Thus, activation of T cells under limiting nutritional conditions can promote long-term tolerance. Along these lines, it has recently been reported that infectious tolerance is in part maintained via the consumption of essential amino acids and the subsequent inhibition of mTOR (22). Indeed, our finding that pharmacologic inhibition of metabolism with NALA, AICAR, and 2DG can inhibit the production of Th1 cytokines is consistent with the notion that inhibition of metabolism (by either depleting nutrients or failing to up-regulate the metabolic machinery) can directly inhibit T cell function.
The two-signal model provides the framework for understanding the acquired immune response. To elicit full T cell activation, Ag recognition (signal 1) must occur concomitantly with costimulation (signal 2), as in CD28 ligation (1). Furthermore, for Th1 cells, signal 1 in the absence of signal 2 leads to T cell anergy (2). The consequence of TCR signaling in the presence or absence of costimulation has typically been evaluated by the generation of various effector cytokines. TCR engagement in the absence of costimulation leads to markedly diminished IL-2 production, and anergic T cells fail to produce IL-2 upon full stimulation. Thompson and colleagues have proposed that an additional critical role for costimulation is to promote an increase in the metabolic machinery necessary for the enhanced metabolic demand associated with T cell activation (14, 23). For example, CD28 stimulation regulates glucose metabolism that enables T cells to meet the increased energy requirements associated with activation. Importantly, these studies indicate that the increase in T cell metabolism is not merely a consequence of increased activation but rather a necessary step to facilitate activation ("fuel feeds function") (3, 24). In their model (Fig. 5⇓, left and center panels), the ability of costimulation to promote the induction of full activation is dependent on the up-regulation of the necessary metabolic machinery. In studying T cell anergy, we have noted that cytokines that are most dependent on costimulation (e.g., IL-2) are the same cytokines that are inhibited in anergy (25). We hypothesized that anergic T cells would display the same profile of metabolic regulation as cells stimulated with signal 1 alone. As seen in Figs. 1⇑ and 2⇑, amino acid, glucose, and iron metabolism are all similarly regulated by signals 1 and 2 and in anergic and nonanergic T cells. Thus, we propose that anergic T cells are metabolically anergic in that even in the presence of costimulation, they fail to up-regulate the requisite metabolic machinery to support full T cell activation (Fig. 5⇓, right panel).
Anergic T cells are metabolically anergic. Thompson and colleagues have proposed that when a T cell receives signal 1 alone, it fails to fully up-regulate the metabolic machinery necessary to support T cell activation (left panel) (3 ). As such, a major function of costimulation is to facilitate metabolic function necessary to fuel the T cell response. We propose that just as anergic T cells fail to produce IL-2 upon rechallenge with full stimulation (signals 1 plus 2), they also fail to up-regulate the metabolic machinery such as the transferrin receptor, amino acid transporters, and glucose transporters necessary to promote a full effector response (right panel). IL-2 has the ability to activate mTOR; however, in the absence of signal 2 (left panel) or when a cell is anergic (right panel), very little IL-2 is produced. In the presence of costimulation (middle panel) AMPK is modestly activated (see Fig. 4B); however, this is not enough to inhibit mTOR in the setting of full activation. Thus, in the absence of energy, glucose, or amino acids mTOR is inhibited, resulting in the induction of anergy. Likewise, a dearth of these nutrients or the inability to import and metabolize these nutrients can directly inhibit T cell function.
Upon activation, the bioenergetic demands of the T cell increase dramatically (3). Blocking the ability to meet these demands inhibits T cell function (Fig. 3⇑). The leucine analog NALA inhibited T cell function. Previously, NALA had been shown to block proliferation in Jurkat cells by inhibiting mTOR (17). The ability of NALA to not only inhibit T cell function but also promote T cell anergy is thus consistent with our previous findings that TCR engagement in the absence of mTOR activation leads to anergy (11). The ability of 2DG to inhibit T cell activation is consistent with other reports demonstrating the ability of this glucose analog to inhibit T cell function (21). As with NALA, we demonstrate that 2DG can inhibit mTOR function and thus promote anergy. Also, we show that by mimicking energy depletion we can both acutely inhibit T cell function and promote anergy by inhibiting mTOR. These findings are consistent with previous findings demonstrating the ability of AICAR to inhibit IL-2 production in Jurkat T cells (8) and mitigate experimental autoimmune encephalomyelitis (26). On the other hand, recently it has been shown that AMPK is rapidly activated in a Ca2+-dependent fashion upon initial TCR engagement (27). Indeed, we see an increase in AMPK phosphorylation with full T cell activation (Fig. 4⇑B). In light of the ability of AMPK to inhibit mTOR, the significance of the immediate activation of AMPK upon TCR engagement is unclear. Perhaps, the initial burst of AMPK activity regulates energy metabolism independently of mTOR inhibition. Consistent with this is the fact that despite the modest increase in AMPK upon activation with signals 1 plus 2, there did not appear to be a concomitant decrease in mTOR activation (Fig. 4⇑B).
Finally, our studies demonstrate that inhibition of mTOR by targeting diverse upstream signaling pathways can all promote anergy. Previously, our group and others have demonstrated the ability of rapamycin to promote T cell tolerance (5, 11, 12, 28). Our new studies raise the possibility that inhibitors of metabolic pathways might prove to be novel inducers of T cell tolerance. Interestingly, whereas higher doses of the metabolic inhibitors blocked cytokine production, their ability to induce anergy was seen at doses that did not inhibit T cell function. For example, the metabolic inhibitors were able to induce anergy at doses that do not inhibit IL-2 production. It has been shown that IL-2 prevents/reverses anergy in part by activating mTOR (29). In this context our observations are consistent with the notion that (at low doses) the metabolic inhibitors promoted anergy by blocking IL-2-induced mTOR activation.
It is becoming clear that controlling nutrient availability can regulate immune responses. This appears to be particularly true when it comes to amino acids. Mellor and Munn have demonstrated that expression of IDO by macrophages and dendritic cells can inhibit T cell proliferation and function by degrading extracellular tryptophan (30). Mechanistically, the IDO-expressing APCs activate the GCN2 pathway in the responding T cells, which can both inhibit their function and even promote T cell anergy (31). Alternatively, it has been shown that dendritic cells can promote T cell activation by taking up environmental cysteine and providing T cells with the amino acid cysteine (32). It remains to be determined if in vivo-specific mechanisms are induced that regulate immune responses by controlling the availability of glucose. Notably, however, tumors are uniquely adapted to rapid growth in part by optimally competing for nutrients. As such, the immediate tumor microenvironment can be depleted of vital nutrients. The tumor microenvironment has been shown to inhibit T cell function and promote tumor Ag-specific tolerance (33). Thus, by limiting metabolic constituents, tumors may inhibit antitumor responses and induce tumor-specific tolerance.
We thank members of the Powell, Drake, and Pardoll laboratories for technical assistance and reagents.
The authors have no financial conflicts of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported in part by National Institutes of Health Grants R01CA098109 and R01CA14227 and by an Ajinomoto Pilot Project grant.
↵2 Y.Z., G.M.D., and C.F.M. contributed equally to this manuscript.
↵3 Address correspondence and reprint requests to Dr. Jonathan D. Powell, The Sidney-Kimmel Cancer Research Center, The Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD 21231. E-mail address: poweljo{at}jhmi.edu
↵4 Abbreviations used in this paper: mTOR, mammalian target of rapamycin; NALA, N-acetyl-leucine amide; 2DG, 2-deoxyglucose; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; PCC, pigeon cytochrome c; AMPK, AMP-activated protein kinase.
Received October 17, 2008.
Accepted September 7, 2009.
Copyright © 2009 by The American Association of Immunologists, Inc.
Schwartz, R. H.. 1992. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71: 1065-1068.
Schwartz, R. H.. 2003. T cell anergy. Annu. Rev. Immunol. 21: 305-334.
Fox, C. J., P. S. Hammerman, C. B. Thompson. 2005. Fuel feeds function: energy metabolism and the T-cell response. Nat. Rev. Immunol. 5: 844-852.
Frauwirth, K. A., J. L. Riley, M. H. Harris, R. V. Parry, J. C. Rathmell, D. R. Plas, R. L. Elstrom, C. H. June, C. B. Thompson. 2002. The CD28 signaling pathway regulates glucose metabolism. Immunity 16: 769-777.
Zheng, Y., S. L. Collins, M. A. Lutz, A. N. Allen, T. P. Kole, P. E. Zarek, J. D. Powell. 2007. A role for mammalian target of rapamycin in regulating T cell activation versus anergy. J. Immunol. 178: 2163-2170.
Harris, T. E., J. C. Lawrence, Jr. 2003. TOR signaling. Sci. STKE 2003: re15
Tokunaga, C., K. Yoshino, K. Yonezawa. 2004. mTOR integrates amino acid- and energy-sensing pathways. Biochem. Biophys. Res. Commun. 313: 443-446.
Jhun, B. S., Y. T. Oh, J. Y. Lee, Y. Kong, K. S. Yoon, S. S. Kim, H. H. Baik, J. Ha, I. Kang. 2005. AICAR suppresses IL-2 expression through inhibition of GSK-3 phosphorylation and NF-AT activation in Jurkat T cells. Biochem. Biophys. Res. Commun. 332: 339-346.
Powell, J. D., Y. Zheng. 2006. Dissecting the mechanism of T-cell anergy with immunophilin ligands. Curr. Opin. Investig. Drugs 7: 1002-1007.
Allen, A., Y. Zheng, L. Gardner, M. Safford, M. R. Horton, J. D. Powell. 2004. The novel cyclophilin binding compound, sanglifehrin A, disassociates G1 cell cycle arrest from tolerance induction. J. Immunol. 172: 4797-4803.
Powell, J. D., C. G. Lerner, R. H. Schwartz. 1999. Inhibition of cell cycle progression by rapamycin induces T cell clonal anergy even in the presence of costimulation. J. Immunol. 162: 2775-2784.
Vanasek, T. L., A. Khoruts, T. Zell, D. L. Mueller. 2001. Antagonistic roles for CTLA-4 and the mammalian target of rapamycin in the regulation of clonal anergy: enhanced cell cycle progression promotes recall antigen responsiveness. J. Immunol. 167: 5636-5644.
Sabatini, D. M.. 2006. mTOR and cancer: insights into a complex relationship. Nat. Rev. Cancer 6: 729-734.
Frauwirth, K. A., C. B. Thompson. 2004. Regulation of T lymphocyte metabolism. J. Immunol. 172: 4661-4665.
Beverly, B., S. M. Kang, M. J. Lenardo, R. H. Schwartz. 1992. Reversal of in vitro T cell clonal anergy by IL-2 stimulation. Int. Immunol. 4: 661-671.
Suryawan, A., A. Jeyapalan, R. A. Orellana, F. A. Wilson, H. V. Nguyen, T. A. Davis. 2008. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation. Am. J. Physiol. 295: E868-E875.
Hidayat, S., K. Yoshino, C. Tokunaga, K. Hara, M. Matsuo, K. Yonezawa. 2003. Inhibition of amino acid-mTOR signaling by a leucine derivative induces G1 arrest in Jurkat cells. Biochem. Biophys. Res. Commun. 301: 417-423.
Reiter, A. K., D. R. Bolster, S. J. Crozier, S. R. Kimball, L. S. Jefferson. 2005. Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside. Am. J. Physiol. 288: E980-E988.
Hay, N., N. Sonenberg. 2004. Upstream and downstream of mTOR. Genes Dev. 18: 1926-1945.
Kimball, S. R.. 2006. Interaction between the AMP-activated protein kinase and mTOR signaling pathways. Med. Sci. Sports Exerc. 38: 1958-1964.
Cham, C. M., G. Driessens, J. P. O'Keefe, T. F. Gajewski. 2008. Glucose deprivation inhibits multiple key gene expression events and effector functions in CD8+ T cells. Eur. J. Immunol. 38: 2438-2450.
Cobbold, S. P., E. Adams, C. A. Farquhar, K. F. Nolan, D. Howie, K. O. Lui, P. J. Fairchild, A. L. Mellor, D. Ron, H. Waldmann. 2009. Infectious tolerance via the consumption of essential amino acids and mTOR signaling. Proc. Natl. Acad. Sci. USA 106: 12055-12060.
Jacobs, S. R., C. E. Herman, N. J. Maciver, J. A. Wofford, H. L. Wieman, J. J. Hammen, J. C. Rathmell. 2008. Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways. J. Immunol. 180: 4476-4486.
Jones, R. G., C. B. Thompson. 2007. Revving the engine: signal transduction fuels T cell activation. Immunity 27: 173-178.
Powell, J. D.. 2006. The induction and maintenance of T cell anergy. Clin. Immunol. 120: 239-246.
Nath, N., S. Giri, R. Prasad, M. L. Salem, A. K. Singh, I. Singh. 2005. 5-Aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis. J. Immunol. 175: 566-574.
Tamas, P., S. A. Hawley, R. G. Clarke, K. J. Mustard, K. Green, D. G. Hardie, D. A. Cantrell. 2006. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J. Exp. Med. 203: 1665-1670.
Colombetti, S., F. Benigni, V. Basso, A. Mondino. 2002. Clonal anergy is maintained independently of T cell proliferation. J. Immunol. 169: 6178-6186.
Powell, J. D., D. Bruniquel, R. H. Schwartz. 2001. TCR engagement in the absence of cell cycle progression leads to T cell anergy independent of p27(Kip1). Eur. J. Immunol. 31: 3737-3746.
Mellor, A. L., D. H. Munn. 2004. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 4: 762-774.
Munn, D. H., M. D. Sharma, B. Baban, H. P. Harding, Y. Zhang, D. Ron, A. L. Mellor. 2005. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 22: 633-642.
Angelini, G., S. Gardella, M. Ardy, M. R. Ciriolo, G. Filomeni, G. Di Trapani, F. Clarke, R. Sitia, A. Rubartelli. 2002. Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation. Proc. Natl. Acad. Sci. USA 99: 1491-1496.
Gajewski, T. F.. 2007. Failure at the effector phase: immune barriers at the level of the melanoma tumor microenvironment. Clin. Cancer Res. 13: 5256-5261.
The Journal of Immunology
Thank you for your interest in spreading the word about The Journal of Immunology.
You are going to email the following Anergic T Cells Are Metabolically Anergic
Message Subject (Your Name) has forwarded a page to you from The Journal of Immunology
Message Body (Your Name) thought you would like to see this page from the The Journal of Immunology web site.
Yan Zheng, Greg M. Delgoffe, Christian F. Meyer, Waipan Chan, Jonathan D. Powell
The Journal of Immunology November 15, 2009, 183 (10) 6095-6101; DOI: 10.4049/jimmunol.0803510
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION
FAR 889
Print ISSN 0022-1767 Online ISSN 1550-6606


text:
On Political Violence
The shootings in Arizona were a terrible thing, no doubt about it. I'm tsking along with everybody else. But here's the thing.
Government is the institutionalization of violence, and democracy is the institutionalization of internal power struggles among interest groups. We are fed ideas about the splendor and glory of its rituals, buildings, and documents, but the underlying foundation remains what it is.
As something of an ex-anarchist, I recognize that institutionalization of violence is on the whole much better than free-floating, chaotic, unconstrained violence, which seems to be the alternative until the messiah comes and the lion lies down with the lamb. So by all means, let us have the best government we can manage, let us keep our political discourse polite, let us try and work out our conflicts with words and ballots rather than with fists and guns. But let us not be surprised when those efforts occasionally fail, because power, coercion, and violence underlie the whole elaborate machinery and sometimes the gears and wires poke through the surface.
The way things are shaking out in the US political system is that one side is willing and eager to deploy the rhetoric of violence while the other side mostly does not (despite that once in office they are just as willing to deploy actual violence). One side is rural and barbaric, the other is cosmopolitan and slick. One side celebrates raw power, while the other likes to pretend it doesn't exist. I'm culturally in the latter camp of course. I want to live in a peaceful, rational, and sane world. But in some twisted and perverse way I appreciate the existence of the former, because while the Democrats like to sweep power and madness under the rug, the Republicans gleefully turn the rug over and make all that stuff visible again.
[[update: Here's someone applying Bruno Latour to the analysis of agency in this situation. As is typical of my reaction to Latourianisms, I can't decide if it's deeply profound or blindingly obvious.]]
[[update: Political violence is as American as apple pie, says a history professor apparently actually named "LaFantasie"]]
Labels: violence
David Xavier said...
Leaving aside your crude hypocritcal partisan reductionism. After all , what more can be said when you start describing people as 'barbarian' hillbillys who are against a "peaceful, rational, and sane world". Well , after what President Bush had to endure from the 'slick cosmopolitans' I think finding rhetorical violence depends on the eye of the beholder....just as what a 'peaceful rational sane world' consists of and how we get there is. By dealing in caricatures to marginalise one group of citizens,allows one to avoid dealing with oppostion to one's ideological world veiw and the investment one has made in imposing it on everybody else.
In the Fort Hood mass killing , the motivations of the gunman were what? The religious ideology that we dare not speak its name? The connections were pretty strong , and the religious ideology in question is "willing and eager to deploy the rhetoric of violence". Of course , we were told not to jump to conclusions about the guman's motivations , and this was echoed in the media quite strongly.
In Arizona , we have an obviously mentally ill person who is talking about mind control through poor grammar. Who ranted against politics in general , whose Congress woman happened to be a Democrat, who happened to be in close proximity in a public space which provided an opportunity for the mentally ill person to act out. Should we jump to conclusions?... hell yeah , it was those rural teabaggin' barbarians and Sarah Palin deploying rhetoric violence. Dont need evidence of any connections or linkages because 'slick cosmopolitans' which , by definiton includes the majority of the media.. can draw these conclusions out of thin air...because you have your caricatures and because you want your version of a " peaceful , rational and sane" world. To achieve such a vision the 'other side' must be de-humanised and de-legitimised by any means possible ... they are, after all, only rural barbarians who pursue madness and power .... gleefully at that.
TGGP said...
Radley Balko says something similar, but with more specific examples, as does IOZ.
The shooter doesn't seem to be a gear/wire poking through the surface but just an out-there pothead. Just as the extremely depressed can't muster the will to kill themselves, what I've heard about him makes it surprising he carried it out. Better watch out for TimeCube guy.
mr waggish said...
http://www.jesusradicals.com/wp-content/uploads/warmaking.pdf
mtraven said...
Thanks for the pointer, but we're on that already.
fsascott said...
You write "...one side is willing and eager to deploy the rhetoric of violence while the other side mostly does not... One side is rural and barbaric, the other is cosmopolitan and slick."
Your notion that the right has a near-monopoly on violent rhetoric is easily exploded by examining what liberals have said about George W. Bush, Dick Cheney, Rush Limbaugh, Sarah Palin, the Tea Party, etc. I could quote hundreds of examples. Here are just a few:
"You guys see Live and Let Die, the great Bond film with Yaphet Kotto as the bad guy, Mr. Big? In the end they jam a big CO2 pellet in his face and he blew up. I have to tell you, Rush Limbaugh is looking more and more like Mr. Big, and at some point somebody's going to jam a CO2 pellet into his head and he's going to explode like a giant blimp. That day may come. Not yet. But we'll be there to watch. I think he's Mr. Big, I think Yaphet Kotto. Are you watching, Rush?" - Chris Matthews, on MSNBC, Oct. 13, 2009
"I have zero doubt that if Dick Cheney was not in power, people wouldn't be dying needlessly tomorrow... I'm just saying if he did die, other people, more people would live. That's a fact." - Bill Maher, regretting that a 2007 assassination attempt on Cheney did not succeed.
"...And then there's Rumsfeld who said of Iraq, 'We have our good days and our bad days.' We should put this S.O.B. up against a wall and say 'This is one of our bad days' and pull the trigger." - fund-raising ad put out by the St. Petersburg Democratic Club, 2004.
More can be supplied upon request.
Democratic map with bullseyes on Republican districts:
http://www.verumserum.com/?p=13647
Leftist attempts to shoot up school board meeting, then shoots himself:
It is striking how such examples never get the publicity that those do which can, however implausibly, be used to suggest that the right has a near-monopoly on violent rhetoric and political violence.
Thanks for utterly missing the point.
I understand your point well enough. But you indeed wrote, didn't you, that "one side is willing and eager to deploy the rhetoric of violence while the other side mostly does not..." This is plainly not true, and these examples are offered in refutation of that statement.
Here's another: the Daily Kos placed a bull's-eye on Gabrielle Giffords back in June of 2008:
http://hillbuzz.files.wordpress.com2011/01/screen-shot-2011-01-08-at-3-05-33-pm.png?w=510&h-384
It is opportunistic charlatanry of the flimsiest tissue to blame the Giffords shooting on some sort of peculiar right-wing attachment to violent rhetoric and imagery. There is plenty of it on the left as well.
I agree with you that government rests upon violence, and operates by it or its threat regardless of who may be in charge. It's the nature of the beast, and futile to hope this will ever be otherwise. That is why I believe in limiting government's scope and authority.
Mexican Chocolate Gelato
Performing Ourselves
The Desirability of Being Maladjusted
Columnists White People Like
The War on Grammar
Not Everything is Free

redpajama_set_name: RedPajamaCommonCrawl


Guide to the Theodore Ole Osmundson Papers,1979-1980.
Theodore Ole Osmundson papers, 1979-1980.
Two articles by Osmundson published in LANDSCAPE ARCHITECTURE magazine.
Member, International Fraternity of Lambda Alpha, Golden Gate Chapter.
Two articles by Osmundson published in LANDSCAPE ARCHITECTURE magazine: "Engineers Relax Controls to Get a Living Levee," November 1980; and "The Changing Technique of Roof Garden Design," September 1979.
Lambda Alpha International. Golden Gate Chapter.
Theodore Ole Osmundson Papers, #3597. Division of Rare and Manuscript Collections, Cornell University Library.


Precisely what is a URL Redirector and just how does URL redirection function?
URL redirection enables you to redirect site visitors from one website to another. In contrast to redirecting a domain name by parking it when the domain is unusable, a URL redirection is possible provided that the domain address is hosted, and thus you shall still have the ability to create 100% functional subdomains and email addresses or even to have content for the domain address in question, though it shall not accessible directly. If you already have a website, for example, but you want to launch one or many localized Internet sites for different towns or countries, you'll be able to work on them without a problem. If a site visitor opens any one of the new domains, however, they will be redirected to the existing website. In this way, you'll not miss customers while developing the localized sites.
When you host your Internet sites with our company and you have a cloud hosting package deal, you'll be able to use our URL redirection tool to forward the visitors from any domain name and subdomain, or from a subfolder under any of them, to an alternative Internet address. The procedure takes a number of simple steps through an easy-to-use interface, so you could set up a redirection even if you don't have any previous experience. You'll only have to pick a domain or a subdomain through a drop-down list, to choose the folder where the redirection shall be set up (the root folder or a subfolder), and then to enter the URL where the website visitors should be forwarded to. For more experienced users, there are also options to select the redirection type (permanent or temporary) and the method (direct or match). Any redirection which you create can be deactivated from the exact same section of the Control Panel, in case you do not require it anymore.
When you create a semi-dedicated server account with our company and you would like to forward any one of your domain names or subdomains, you can use the practical redirection tool we have incorporated with our custom Hepsia hosting CP. It'll enable you to forward the traffic within seconds, because all you will have to do is pick a domain/subdomain and type the web address of the other site. The forwarding shall take effect right away. If you are experienced, you'll be able to modify different options, including the type of the forwarding - temporary or permanent, and the method - direct or match. These options can be modified for any already present redirection also, so you will not have to create a new one if you want to modify something. You may remove a redirection by clicking on the Delete button associated with it.


We were instructed by Mayfair Capital on behalf of their PITCH Fund to dispose of Omron, an attractive 22,634 sq ft headquarters office building in out-of-town Milton Keynes.
The tenant re-geared their lease for a further 12 years in August 2018 at a passing rent of £325,000 per annum (£14.36 per sq ft) with an upcoming rent review in year five, providing an attractive opportunity to investors seeking to capitalise on the recent rental growth in Milton Keynes, a result of a lack of stock and country-leading economic growth rates.
We undertook detailed pre-sale due diligence and undertook a full brochure-led PR and marketing campaign, attracting a robust level of investor interest including a number of offers at or above the asking price of £4.35m.
The property was sold in January 2019 at a price of £4.735m, reflecting a net initial yield of 6.44%, significantly higher than the asking price to a joint venture vehicle of two eastern county councils.
Hexagon House, Milton Keynes: disposal of out-of-town office building Our work Instructed by Screen GP Europe (BV).


From one of the world's most recognized experts on management comes a simple parable filled with insights designed to help readers manage change quickly and prevail in changing times. Written for all ages, the story takes less than an hour to read, but its unique insights can last for a lifetime.
Patrick Spencer Johnson was born in Watertown, South Dakota on November 24, 1938. He received a bachelor's degree in psychology at the University of Southern California and then graduated from the Royal College of Surgeons in Ireland. While working in a hospital, he grew frustrated at seeing the same patients return with the same ailments, as if they were not trying to get better. He left the hospital to work as director of communications for Medtronic, a medical device manufacturer. He wrote short books to help customers understand complicated technical information. He went on to write short books about life and business including The One Minute Manager written with Ken Blanchard, The Precious Present, and Who Moved My Cheese? He died from complications of pancreatic cancer on July 3, 2017 at the age of 78.


text:
Man, 77, jailed for 10 years after being convicted of rape and indecent assault
A man has been jailed for 10 years after he was convicted of rape and indecent assault of a child.
David Thomas, 77, from Aldershot Road, Fleet, was sentenced at Salisbury Crown Court on Friday 29 April following a week-long trial.
He was convicted of rape and indecent assault of a girl aged under 16. Thomas was found not guilty of another count of indecent assault.
The offences took place in Farnborough between 1984 and 1988. It was reported to police in 2020.
Thomas was sentenced to 10 years in prison on Friday.
Detective Constable Michelle Carrig, from Operation Amberstone North, said: "I hope this sentence has provided the victim with some element of closure, knowing that Thomas has been sentenced for a very long time.
"Thomas committed harrowing crimes against the victim and I would like to praise her for showing courage by coming forward and contacting the police.
"We take incidents like this extremely seriously and I would like to encourage any other victims, or anyone who has been a victim of sexual abuse, or if they know someone who has been impacted by sexual abuse, to contact police on 101."
As a survivor of sexual abuse, you are entitled to support whether you report the crime or not.
You can access different types of support depending on how you are feeling and what you decide to do next.
You can visit https://www.hampshire.police.uk/advice/advice-and-information/rsa/rape-and-sexual-assault/support-for-victims-of-rape-and-sexual-assault/ to see the support on offer.


In a space of starting to be curiosity to veterinarians, the authors have produced a swift connection with the sensible medical elements of small animal dentistry. The textual content is prepared to mirror the clinician's considering and method of difficulties: heritage info, scientific relevance, key issues, differential diagnoses, diagnostic exams, and administration. a few four hundred most beneficial illustrations—color images, imaging and diagrams—provide a seriously very important supplement to the text.
The colour handbook—here with revised textual content and references—offers real-life insights into the development of oral sickness and has been welcomed as a operating source by way of veterinary practitioners and scholars, and as a useful assessment by means of extra complex veterinary dentists.
Now in complete colour, with over 1,400 photos and illustrations, the 6th version of this market-leading textual content is a step by step, hugely visible consultant to the how-to's of periodontal instrumentation. It takes scholars from simple talents akin to sufferer positioning, intraoral finger rests, and uncomplicated instrumentation, all of the strategy to complex suggestions similar to evaluation of periodontal sufferers and instrumentation of multirooted tooth, root concavities, and furcation parts.
PDQ Oral disorder: prognosis and therapy is designed to function a major resource for the identity of illnesses and prerequisites of the mouth and jaws. Dentists and physicians can with no trouble entry a large spectrum of entities with short, crucial textual content accompanying consultant medical images and radiographs.
Getting into its 3rd version, the bestselling guide of organic results of Electromagnetic Fields is a definitive reference for getting to know bioeffects of static, low-, and high-frequency fields. It offers a well-rounded standpoint at the organic results of electromagnetic (EM) fields, authored via lively members to the sector with a wide selection of pursuits and backgrounds, techniques, and interpretations.
Computational and Evolutionary research of HIV Molecular Sequences is for all researchers drawn to HIV examine, even those that in simple terms have a nodding acquaintance with computational biology (or people who are conversant in a few, yet no longer all, elements of the field). HIV study is rare in that it brings jointly scientists from quite a lot of disciplines: clinicians, pathologists, immunologists, epidemiologists, virologists, computational biologists, structural biologists, evolutionary biologists, statisticians and mathematicians.
These two compartments do not communicate with each other. A thick band of fibrous tissue on the lateral aspect of the joint capsule forms the lateral ligament which tightens when the jaw opens18,21 The TMJ is a condylar joint that can move in flexion, extension, and translation. Translation refers to the mandible's ability to move rostrally and laterally. The degree of translational ability is related to the dietary habits of the animal. A strictly carnivorous animal will have less movement rostrally and laterally than an omnivore21–23.
Lymph node staging of the primary neoplasm is an integral component of the multimodality treatment plan for the cancer patient. • Evaluation of the mandibular lymph node alone in oral cancer patients may not provide a true indication of metastasis. 5% of cases with metastatic disease to regional lymph nodes have metastasis to the mandibular lymph node31. Tonsils may normally be in or out of their tonsillar crypt. (1: Crypt of tonsil; 2: Soft palate; 3:Tonsil [out of crypt]) CHAPTER 2 39 Oral Examination Lee Jane Huffman • Step 1: History • Step 2: General physical examination • Step 3: Orofacial examination • Step 4: Conscious (awake) intraoral examination • Step 5: The anesthetized orodental examination 40 CHAPTER 2 Oral Examination Step 1: History Oral examination is prefaced by obtaining and verifying signalment and patient history while listening to the client, observing the patient, and performing the complete physical examination.
Infiltrative lymphadenopathy). • Palpate the thyroid glands. • Assess the position of the globes and ability to retropulse them. g. polyps; infections; extension of neoplasia). g. noting dander or keratin plugs, erythema, alopecia, lumps, or bumps. • Examine for draining tracts. Any tracts should be sterilely swabbed (for cytology, culture-sensitivity) and gently pressed upon to assess pain, volume, and consistency of fluid. KEY POINTS • The first step of orofacial examination is external orofacial survey examination, which is performed without exogenous opening of the patient's mouth.


text:
DUNGEONBOWL RULES PDF
02.01.2019 02.12.2020 admin Science
Necromunda Dungeonbowl Basic Rules There have been two official sets of Dungeonbowl rules published by Games Workshop: The original rules as included in the Dungeonbowl boxed expansion of , and an update of the Dungeonbowl rules for the 3rd edition of Blood Bowl, published in White Dwarf Magazine in A summary of these basic rules can be found below. All the rules presented on these pages have been written to function with the 1. Dungeonbowl Essentials The essence around which Dungeonbowl as a Blood Bowl game variant revolves can be summed up relatively easily, since it is basically a method of setting up an alternative game board, and some very simple rules for how the players interact with the special features of this alternative board.
Author: Doujas Akirr
Uploader: Faegor
These core rules are summarised below. Setting up the Game The first thing to do is for the players or a GM to set up the dungeon in any mutually agreeable fashion.
It must contain one end zone per team, placed an appropriate distance apart, and at least six chests and six teleporters. The teleporters should be numbered so that you can select a random teleporter by rolling a die. The chests should either use double-sided tokens that show one of the chests to have the ball and the rest to explode, or you should randomly roll each time a chest is opened to see if it contains the ball. When starting the game, each coach may set up up to six players in their own end zone.
Do not roll on the Kick-Off Table when starting the game. Once per turn, a coach may activate one of his reserve players from the dug-out and teleport them into the dungeon and onto a randomly chosen teleporter. Alternate player turns following the normal Blood Bowl rules, and continue playing until one player scores a touchdown and wins the game. Any adjacent player may open a chest at the cost of one square of movement.
If the chest does not contain the ball, the chest explodes ad all adjacent players are knocked down. After a chest is opened, it is removed and the non-exploding chest is replaced with the ball. An unopened chest counts as a square containing a prone player, and can be leaped over. Teleporters: Teleportation happens automatically as soon as a player steps or is pushed onto a teleporter, after which they are instantly moved to one of the other teleporters in the dungeon as indicated by a dice roll.
If the player rolls the number of the teleporter they are already on, they are lost in space. Players that are lost in space are unharmed, but are removed from play for the rest of the game. If they teleport onto a teleporter that already contains another player, that player is instantly re-teleported, and this chain must continue until all teleporting players have either landed on an unoccupied teleporter or are lost in space. If a player tries to teleport more than once during the same action, the player is automatically injured.
Passing: The ball may not be thrown to more than short pass range in the dungeon. Instead of throwing at a specific square, a player may choose to throw the ball into a wall in order to get it to bounce back at an angle. To do this, the player nominates a square next to a wall and makes a pass as normal. If the pass is accurate, the ball hits the nominated wall square, if it is inaccurate, it either hits the square to the left or right roll a D6.
Once the hit square is found, treat the ball as if it was thrown in by the crowd, as it bounces back 2D6 squares in a direction as indicated by the throw-in template. If the ball hits another wall before it has run out of squares, it ricochets in a 45 degree angle off the wall in the opposite direction that it came from, or directly back if it was directly aligned at a 90 degree angle.
Once it has run out of movement, it stops and bounces once. Last updated in March
JRCALC 2013 PDF
Necromunda Dungeonbowl Expanded Rules The Dungeonbowl: Astrogranite Edition rules that expand on the basic rules cover a variety of subjects, supplementing almost every aspect of the game. The expanded rules can be divided into the following subsections. Base Rule Modifications and Additions : This section gives a selection of rules that directly modify or supplement the basic rules of Dungeonbowl and Blood Bowl, i. Dungeon Features and Variables : This section is all about how to make a Dungeonbowl dungeon more interesting. This includes ideas and rules for how to make your dungeon multi-level, and how to add features like traps, water, and chasms to your rooms and corridors.
KABULIWALA BOOK PDF
Dungeonbowl: Rules
Now, this is where it gets a little esoteric. Sometimes, the game will not automatically select the best path for you. During his turn a coach may move his players as well as make a block with those who are in direct contact with an opponent. If the player rolls the number of the teleporter they are already on, they are lost in space. LRB6 DungeonBowl? Rainbow gets Halfings, Humans and Elves. You will receive a -1 penalty on your Agility roll for each opposing player within one square of the ball.
PASS4SURE CCNA 200-120 PDF
Facebook Background Dungeonbowl, as its name suggests, is played in a dungeon and is a variant of the mighty popular Blood Bowl! The teams move about using teleporters, and must begin the game by searching for the ball hidden in one of the trapped chests scattered throughout the dungeon. The Dungeonbowl league is sponsored by the ten Colleges of Magic, and each College also supports one of the teams taking part in the tournament. Even though the idea was originally to settle the matter of which College was the most powerful, and was to be a one-off event, Dungeonbowl has now been going for over 20 years and shows no sign of ending in the foreseeable future.
GUARDERAS SEMIOLOGIA PDF
INTRODUCTION TO TPM BY SEIICHI NAKAJIMA PDF
COMPORTAMIENTO DEL CONSUMIDOR LEON G SCHIFFMAN PDF
DEMOKRATIK ANON PDF
BAAL E JIBREEL PDF
ACTA ORNITHOLOGICA PDF
CONVERT FLOWDOCUMENT TO PDF
ASTM C373-88 PDF
DE MYSTERIIS AEGYPTIORUM PDF


Recruitment, management and payment of seagoing personnel.
Since 2010, MN took over Morlenn Express and its activity of maritime transportation of French Navy personnel betwenn its on Brest roads. For doing so, Morlenn Express owns 6 launch boats : Bindi, Tibidy, Trébéron, Arun, Terenez and Louarn which can as well be used occasionally on other activities.


Hello and welcome to the KNAUS fan shop.
We have compiled a wide range of large and smaller items to make travelling even more enjoyable. From high-quality ladies' and men's fashion to key rings, there is something for everyone.
A great deal more merchandise coming soon!


We are a leading international service provider to the oil and gas production and processing industry. Our teams design and build oil and gas infrastructure; operate, maintain and manage assets; and train personnel.
We have a 37-year track record and have grown significantly to become a constituent of the FTSE 250 Index, with 31 offices and around 12,750 staff worldwide, comprising more than 80 nationalities.


5 Acres & A Dream: The Big Question: Have We Grown & Stored Enough???
The Big Question: Have We Grown & Stored Enough???
Continuing from my last post, Jewels of the Summer Harvest, the question about my food storage is, have I grown and stored enough to feed us until the next harvest? Our ultimate goal is to be food self-sufficient. Realistically, this has to be taken in steps, so my food preservation goal for 2010, focused on only one food category, to grow and preserve all the vegetables we would need.
In this post, I've looked at that and a little more. This is an important analysis, because it will help me determine how much to plant next year. Of course, it's hard to pick a starting point of when we'll dip into our stores. Something like pizza sauce, I'll start using as soon as I run out of the previous year's. Things like dinner vegetables aren't so clear cut. If I have something fresh growing in the garden, I prefer to use that. But if we get a hankering for sautéd okra, then I'll get some out of the freezer regardless of what else is growing in the garden.
With all that in mind, I've tried to estimate our needs by food categories: fruit, vegetables, and meat, but also things like jams & jellies, soups, sauces, and starches. In the past, I simply used 52 weeks as a basis for this. For example, if we wanted to eat green beans once per week, then I needed to can 52 quarts of green beans. This time, I tried to take into consideration the fact that during summer months, we will be eating fresh from the garden. Also, I'm learning to overwinter root vegetables in the garden itself, so these don't need to be stored, since they are available as long as the ground isn't frozen. Someday we'll add a greenhouse, and that will change storage needs again.
These estimates are approximate minimal needs for just the two of us. That's my starting point. Of course, I'd like to have more for the times we are feeding more. Or, for the times when a certain crop doesn't do very well. For example, my sweet corn didn't do well this summer, so all I got for food storage was one pint to freeze. But again, these are things I'm working toward. Things that will have to be taken in steps.
have in storage: 92.5 quarts + winter squashes (one per meal) = 99 quarts worth in the pantry so far, plus broccoli, which hasn't been harvested yet, and turnips, beets, and carrots which I can overwinter under mulch in the garden.
This one's tougher. If I can get three meals from one chicken for the two of us, then our 11 chickens will last us for 33 meals. Since DH likes meat for dinner, that's obviously not nearly enough. We're already planning on raising a big batch of meat chickens next year. We're also discussing more soups and stews, which will stretch our supply out some. He'll accept eggs, however, we usually use these for breakfast, lunch, and baking. I can go through 2 dozen eggs a week easily, so my 12 frozen dozen won't last but a month and a half during non-laying season. We talk about substituting complete vegetable proteins, for example the black turtle beans with whole grains, but he still misses his meat. This is where saving fats from bacon and sausages, and bones helps. If I sauté my onions and veggies in these fats, or cook soup or beans with bones, it gives the dish a meaty flavor, and that helps. I have over a gallon of dried black turtle beans, so there is a lot of soup and bean dishes. We're discussing getting a couple of pigs next year, also raising a couple meat goats. The question here though, is are we ready to butcher more than just chickens???
I do purchase locally pasture raised beef and pork. These are a bit more expensive, but with the money I save from growing our own fruits and vegetables, we have extra dollars in our food budget for this.
Soups - I love soup for lunch, and make a lot of soup from leftovers, dried beans (of which I have over 2 gallons of homegrown dried black turtle beans), or split peas (purchased in bulk). That makes it hard to calculate how much canned or frozen soup I really need. It's just nice to have the convenience of ready-to-heat soups on hand. I'm not even going to try to guess how much I need. I'll just make a note of when I run out, and plan for more next year.
have: 16 quarts. Will probably end up saving these for shopping and errand days.
have - 26.5 pints. That's just squeaking by, isn't it!
have - 13 pints. Obviously need to plant more cukes! This doesn't include pickled beets, which have yet to be sampled.
Starches - all meals, year round. So far we've only grown potatoes in this category. We harvested in July and 3 months later, out of 125 pounds, we have about 22 pounds left (some lost during storage, some made into soup, quite a few eaten.) We still have fall potatoes yet to be harvested. That's an important dinner starch, but I also need to consider flour for bread and baked goods. I'm planning an experimental patch of winter wheat soon, and next year we'll try to grow field corn.
Eggs - included with proteins above, but if I try to consider what I'll need on a weekly basis, I'd say we can go through 2 dozen easily. I have 12 dozen frozen, so that's 6 weeks worth. Not sure how long my hens will lay, nor how egg production will decrease as they get older, so I still have some experimenting to do before I can calculate this one with at least a small amount of accuracy.
In addition, I have frozen broth; dessert ingredients like pie fillings; ingredients like dried onions and frozen green peppers; dried fruits I'll use for baking such as figs, bananas, and watermelons; pickle relish; enough pumpkin for 2 holiday pies; dehydrated tomatoes, pesto, and herbs. Bulk purchases include beans, grains, flours, and meat.
So. To answer my own question, have we grown and stored enough? In terms of my first short term goal, yes. For this year, it was to grow and preserve all our vegetable needs. On that score, we've been successful both with dinner vegetables and pizza sauce. As a bonus, we're still getting fresh veggies from the garden and will have root crops in the ground. For some things I'll have to wait and see, for example, how long the frozen green peppers and dried onions last. Next year I can make adjustments accordingly. My bonus category is jams and jellies. Hopefully we do have just enough till strawberry season comes around in the spring.
Other categories remain as future goals, meat and starches for example. Even though we don't have a year's worth of potatoes, I've learned a lot and can plan for more next year. Meat self-sufficiency will take longer to reach, as it requires preparation for the animals, and the ability to feed them too. Grains too, as we need to learn how to grow and harvest them. Fruits are waiting on our fruit trees to mature. The things I didn't mention are sweeteners and fats. In the future we'll have bees, and if we do get pigs, we'll have some of the fats too. It's one step at a time, and I'm happy with how far we've journeyed to date.
In reality, this is a lot of guesswork. It is one of those issues which is best answered experientially. Since this is the first year (in this place and with this garden) that I'm making a deliberate effort in this direction, I won't actually know how accurate I've been until this time next year, I'll have a better idea of when I ran out of certain items, and whether or not I'll need to plant more. One step at a time. I can't say that often enough.
Your turn. How would you calculate food needs for your family?
What an awesome inspiring list. I am wanting to lean more about winter/ roots in the garden. Can you reccoment a book or website? Love the list of actuals and needed. Good job!
Melissa, thank you! It is so encouraging any time you all are inspired by something I write.
Good question about books or websites, but I honestly don't know of any specific to the topic. I just picked up tidbits of information here and there, and started experimenting.
The gist of it is this: that cool weather root crops (like parsnips, carrots, beets, and rutabagas) will survive in the ground and remain accessible as long as the ground isn't too frozen to dig them out. A 6 - 8 inch layer of mulch is recommended to help protect the ground from freezing. I think this works because they're biennials, and are waiting to grow again in the spring.
Another way to store them in the ground, is to dig a trench or pit, line it with straw or leaves, put the roots in, and mulch well. There is a book, Preserving Food Without Freezing or Canning, that shows several ways to do this. It's a great book because it has a lot of other "forgotten" food storage techniques as well.
Toodie, you are touching on a really important concept, that of community. You explained it very well. I do hope you blog about it! It should be a goal for all of us, really.
I agree with the others, very impressive. though not possible for us:(( we don't have enough land to consider growing all our veggie foods (incl. "starch"). and we don't have the climate to be truly successful with grains. never mind the land we'd need to keep our own cattle and pigs. it's just not feasible, all we can do is try to supplement our vegetable needs as much as possible. I don't think I'd be able to slaughter pigs though. the animals are quite large and you do need a lot of space to slaughter - and afterwards to prepare all the meat. apart from the fact that we still shy away from killing chicken - never mind a pig:)) I know, we're cowards.... but looking at your numbers I'd say you're already pretty good in achieving your goals - considering that you've only started with this a year ago!
I think in this day and age it would take some relearning to be able to do this. I've grown up as an X generation grocery store shopper. X meaning I have no idea how long it's been since anyone in my family history has tried to do this.
I'm so looking forward to see how you do this year.
I see that I'm not the first person to be inspired by this list!!
Leigh, do you eat many nuts? Do you have the potential to produce your own? Also, what will you plan to do for milling grains? Part of me is very keen to use a quern stone - the other part of me is very aware of the damaged teeth I've seen from archaeological digs. Damaged, because the bread the people ate every day contained so much grit it actively eroded their teeth! Not so fun.
I have the same problem as Alison... but times two! I have two picky eaters who will like something and then all the sudden change their mind without telling me the shopper. sigh!
I was coming back to tell you that I sit down with a calendar to plan out our menu...making sure to note any activities that might mean we need a quick dinner. Then I usually make our meals so that we have left-overs (plan overs) for the next day...I cook on one day, and take care of the dishwasher on the other day. However, we always end up with something that messes that all up. If I had to plan out meals for a year I don't know how I could do it.
Renee, that's exactly right and it's the biggest challenge Dan and I face. So many practices that were common once are nearly forgotten. We are having to learn new ways of doing things as well as accept our geological limitations. It isn't often easy. Sometimes we encounter folks for whom the concept is so foreign, that they think we're totally off our rockers!
I thought it was interesting how you plan your food needs. We almost approach it from opposite directions. You plan the menu and shop according. I just buy or grow things we like and then figure out each meal a day or two at a time. It would definitely be a challenge to have picky eaters like you and Alison do.
Alison, yes to growing nuts. We have several pecan trees, a hickory tree, and have planted an almond tree. Nuts is one thing Dan is fussy about, so I don't think about trying to plant more.
You are so amazing! I'm aiming for the same things, but have only about 1/3 acre and there are 4 of us. Some things are easier than others. Great job!
I've just spent the last hour+ enjoying your homesteading adventures. You've brought back many memories for me, of my 20's when I was a "back to the land" kind of gal, complete with wood cook stove and no electricity nor indoor plumbing even.
There is, indeed, a lot of work and planning that must be done, but your "jewels" really are priceless. One word: YUM!
I'm just now getting back into my own daily rhythm and have been getting in spinning, but not yet much weaving. That's about to change in the next couple of days as I plan and wind a new warp.
Am looking forward now to each new installment. Now, I'm off to explore some of your links.
It'll take a couple of years growing and using up the food - and running out. I'm so impressed, though, with what you've done, both the accomplishments and the preparations! You're an inspiration!
Heather, you know, I think we all struggle somewhat with the amount of land we have. Sometimes our 5 acres doesn't seem like enough (like the days when Dan wants a horse), other days it seems like we will never be able to tend to it all. Some areas the weeds are out of control and I can't seem to find the time to conquer them. The folks I'm really impressed with are the Dervaes (Little Homestead in the City).The amount of food they produce on 1/10th of an acre astounds me.
Jane! I am so happy to hear from you! I've thought about you often, wondered how you're doing, and waiting patiently until you start blogging again. Sounds like our pasts are something else we have in common, LOL.
Katrien, that's what I figure too. It will all boil down to experience in the end. I'm finally figuring out what will help me most will be to record the weight of seed planted, how much produce I was able to put by from it, and how long it lasted us. For the time being, I'm just putting up everything I can and keeping track of it.
Leigh, I've said it a bunch but I'm going to say it again....I love your blog. I can so relate to what ShuttlePilot said because that is exactly what I did when I first found your blog and this most recent post continues to amaze and inspire. I will be reading faithfully to see how you make out with all your food stores.
Alison, sorry. I answered about the nut trees, but failed to mention that yes, I do have a grain mill. It's hand operated, but it has excellent torque and makes fine flour. I'm hoping to get it set up soon, and will post about that too.
Vicki, I truly appreciate that. Comments like yours and Jane's inspire me too, to press on, to overcome, to keep on writing and recording. Toodie mentioned a community to share and barter; that's what we are on the internet too. A community to encourage one another and share ideas. As Renee said, so much of this knowledge has been forgotten. We learn from one another.
I was looking at your last post and remembered how much pride my mom had in her canning room in our basement. The shelvers were full and having a family of five come spring we used every bit of what she canned. Since we were potato farmers we had as many of those as we wanted in the warehouse in town.
Your set up is very impressive!!!
For "Guesswork" you have done a lot of thinking and calculating on this subject.
And you are thinking about your future needs, too, which shows great planning. Getting pigs is a good idea, they are walking garbage disposals that turn that food garbage and left overs into wonderful meat for the winter.
And meat chickens, too. I remember that one year we butchered nearly 100 chickens and that fed us and my grandparents for the year, and this was on top of the half beef and half hog we had taken to the locker plant.
I am impressed at what you have accomplished.
You are quite the little industrialist ant aren't ya? We butcher and keep the freezers of us and our two children's freezers filled. I really don't put up many veggies. I know...my bad. I can purchase the frozen so cheap and I spend all my energy on my flower gardens. My dear MIL always put out a veggie garden big enough to feed the whole dang country!
God bless ya and have a incredible day sweetie!!!
Barb, I think there is just something fulfilling about having a work from our hands. Whether it's gardening and canning, knitting, sewing, weaving, furniture making, whatever; there's a sense of fulfillment in having something to show for what we do.
We're thinking that getting a pig during the summer would be fairly inexpensive to feed, considering the amount of vegetable "waste" we have from gardening and canning. Not sure if we'll be prepared this spring, but the one after that for sure.
Nezzy, I'm trying! Beef is one thing I can't see us raising on our own. Not enough pasture for one thing. Still, meat is the biggest expense, so being able to butcher and store all one needed would be a huge blessing.
Carrie, thanks! I'm sure there are other ways to approach it. These are the only ones I've figured out. Eventually it ought to become 2nd nature, but that will take a few more years to say the least.
We're doing the same thing - except that, for now, we're not growing our own. HOW do you figure out what you're going to need for an entire year? And then, without the ability to stop time, how do you get it all preserved and stored in time?
I feel like I canned like mad throughout the fall. I have less than 80 pints of meat canned, and only a dozen jars of potatoes. Not a dent in our needs, not even enough to get us through to the summer.
The Preserving Without Canning or Freezing book should be arriving at my door in the next day or so. I was expecting it today.
I'm going to enjoy reading your blog, since you're doing some of the same things I am.
Canadian Doomer, that's the million dollar question, isn't it? I tried to figure what we'd need based on how many times a week we'd want to eat that particular item. The growing part though, is a lot of it is trial and error. In the end, I'll have to wait and see if my estimates were correct. I think it may take several years before we can actually meet all our own vegetable needs. We're learning about raising our own meat too, but feel we need to raise our own feed as well.
Basically I operate on the principle that something is better than nothing. Some garden is better than none. Some food stored is better than none. Right now I'm focusing on learning to save all my own seed as well as preserving everything I can! Preserving Without Canning or Freezing is a great book. I also want to get some Tattler reusable canning lids.


00:32 Property bubble is elsewhere in the world 00:50 Property bubble in the US 03:46 What happened in Australia during global financial crisis? Good day everybody Daimien here from Integrity Property Education. If you are looking for some property investment strategies, advice, information then you come to the right place. Today's blog video is about the "Property Bubble". Is there a property bubble in Australia and is it going to pop? The first thing that we need to understand is that there is a property bubble elsewhere in the world and yes they have popped. The main example is in the US. We did see a similar effect in the UK, because their banking sectors are closely tied together.
What happened in the US was quite interesting. The government over there set up 2 banks and started lending money to the people who the other bank won't lend money to. So these 2 banks were called Freddie Mac and Fannie Mae. They lent money to the people that could normally get loans. That was interesting with good intention, of course, they had wanted every American to be able to realize their American dreams of having their own homes, and so they started lending them money. This time when the idea came up, the property market was very strong and the economy was very strong, so they started lending money to these people.
What did that do to the property market?
What did that do with the property market was it reduced the number of renters and increased the number of buyers. The rents basically started going down and didn't move at all and the buyers went through the roof. That in term, put house prices up. All of these people were able to buy houses that they couldn't buy before and the property market was going up, but then there were started housing problem. Inflation started to take off because there were so many people buying houses and so much money flying around the economy. As the inflation started to go up, the American Reserve Bank had to put interest rates up to slow down the economy. When this happened, it was the beginning of the end. Why is that? Because these people that they lent money to, have these loans called "Non-recourse Loans". Non-recourse loans – basically the only security on the property was the property itself or the only security on the loan was the property itself. If they couldn't afford the mortgage anymore, all they had to do was leave the keys on the door, ring the bank and say "Sorry, I'm Out!" This happened as interest rates went up, all of these people started foreclosing on their mortgages. We went from a situation where we had oversupply of buyers pushing prices up, to under supply of buyers and mortgages foreclose everywhere and it put the mortgage market in the free fall. All of these foreclose flying in the market and house prices tumbling down quite dramatically. Behind all that, there was also this thing called the "Subprime Process". Subprime Process – these mortgages that were linked on the non-recourse were referred to a subprime mortgages. Why is that? Because they are non-recourse, subprime meaning, less than prime. They were not very good securities but they were selling them and trying these mortgages as a security behind the scenes in a complex arrangement of the investment scams. What then happened, when the foreclosure went down and had to claim all these, the security became worthless because house prices dropped below the value of the mortgages and then we had a big crisis that spread across the world and became the global financial crisis.
During all of these, what happened in Australia?
Interest-Only Loans – basically you don't have to pay the money back. You just got to service the interest.
Reverse Mortgages – these where people capitalizing mortgage re-payment by just adding it on top of the loan.
So the banks basically worked out that they make money by charging people interest. So why they wouldn't want you to pay interest to them as long as possible? That is how they make their money. People can borrow more money for a longer period of time, therefore they can borrow more money on the same income and that is why we have this great multiple.
There is no property bubble in Australia ladies and gentlemen and if you think there is and you are stopping yourself from buying a property, well that little obstruction in your mind is really going to disadvantage you financially. You are going to be in to win it! Go out there invest in property make sure you know what you are doing, of course, seek to understand and don't listen to everything you hear in today current affairs because, generally those people aren't experts in the field. They are just regurgitating some other rubbish that tends to sell newspapers or sell advertising space.