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Most tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells.
Methodology/Principal Findings
We used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-γ, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-γ production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR.
These data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2.
Citation: Bendle GM, Xue S-A, Holler A, Stauss HJ (2007) A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function. PLoS ONE 2(4): e353. doi:10.1371/journal.pone.0000353
Academic Editor: Jacques Zimmer, Centre de Recherche Public-Santé, Luxembourg
Received: March 7, 2007; Accepted: March 13, 2007; Published: April 4, 2007
Copyright: © 2007 Bendle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a research grant of the Leukaemia Research Fund (LRF), UK. The LRF was not involved in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript.
Competing interests: HJS is advisor to the start-up company Cell Medica.
Mechanisms of both central and peripheral tolerance have evolved to silence T cells with specificity for self-antigen. Whilst these mechanisms of T cell tolerance reduce the risk of autoimmunity, they also represent an obstacle to successful T cell-based cancer immunotherapy, as most T cell-recognised tumor antigens are also expressed in some normal tissues [1]–[5]. A better understanding of the mechanism and the magnitude of tolerance to tumor-associated antigens (TAAs) will be essential for the rational design of TAA-specific immunotherapy strategies for the treatment of cancer. As the antigen expression pattern in normal tissues is likely to have a major impact on T cell tolerance, it is desirable to develop models that can provide information on how the physiological expression of TAAs in normal tissues affects the development and function of TAA-specific T cells.
In the past T cell receptor (TCR) transgenic models have been used successfully to provide insights into the mechanisms of central and peripheral tolerance. Antigen expression in the thymus results in clonal deletion of developing T cells, although deletion is sometimes incomplete as T cells can escape into the periphery [6], [7]. For antigens that are expressed outside the thymus, antigen encounter in the periphery may result in T cell deletion or the induction of a state of unresponsiveness, often referred to as anergy [8]–[11]. Identified mechanisms that can lead to unresponsiveness include down-regulation of the TCR and accessory molecules such as CD8 co-receptor, or the disruption of signalling pathways required for IL-2 production [6], [10], [12]–[14]. In addition to deletion and anergy, ignorance is a form of tolerance where the expression of antigens in certain immunologically privileged tissues is ignored by peripheral T cells [15]. Whilst ignorant tolerance is easily broken by immunization, deletion is irreversible, and strategies for the reversal of anergy may require some knowledge of the mechanism and the level of T cell unresponsiveness.
Recently, two transgenic mouse lines expressing TCRs specific for two defined TAAs have been developed in order to analyse how TAA expression in normal tissues affects T cell function. In one model, the TCR was specific for an epitope of gp100, a TAA that is over-expressed in most melanomas while its physiological expression is restricted to melanocytes [16]. In another model, the TCR was specific for an epitope in the gag protein of Friend Murine Leukaemia Virus (FMuLV), which is a major CTL target in FMuLV-transformed murine tumors [17]. In this case gag-transgenic mice were generated to target the expression of this model TAA to normal liver tissue. The immunological analysis in these two models revealed a relatively low level of tolerance against gp100, whilst T cell tolerance against gag was more profound. It is possible that these difference were due to differences in the tolerogenic properties of skin melanocytes versus the liver, although it is also possible that high level of transgene driven gag expression may have contributed to a more pronounced T cell tolerance. Here, we set out to assess tolerance to a TAA that is naturally expressed in most normal tissues.
MDM2 is over-expressed in a large number of human malignancies of haematological and epithelial origin [18]–[23]. The MDM2 oncogene functions by inactivating the p53 tumor suppressor protein [24], [25], and its over-expression in tumors was shown to contribute to the process of malignant transformation [18]–[23]. Using HLA-A2 transgenic mice and the allo-restricted strategy to circumvent tolerance to MDM2, we have previously demonstrated that CTL specific for an HLA-A2 presented peptide can kill a panel of human leukemia cell lines, while MDM2 expression levels in normal cells were too low to trigger CTL killing [26]. We also observed that CTL with this specificity were not detectable in HLA-A2 positive humans, which was compatible with the suggestion that tolerance mechanisms may have inactivated or deleted such CTL.
As the MDM2 expression pattern in normal tissues is similar in humans and mice, we generated TCR transgenic mice to study tolerance mechanisms against this TAA. Previously, we isolated from H2d BALB/c mice high avidity allo-restricted CTL clones specific for an MDM2-derived peptide, pMDM100, presented by H2-Kb class I molecules [27], [28]. In this study, the TCR genes from the CTL were isolated and used to produce transgenic H2d mice that did not present the TCR recognised pMDM100 peptide (referred to as Agneg mice), and H2dxb F1 transgenic mice in which the peptide is presented by normal tissues (referred to as Agpos mice). These mice enabled us to determine how physiological MDM2 expression in normal tissues affected the development and function of T cells specific for this TAA.
We found that some MDM2-specific T cells escaped thymic deletion and accumulated in the periphery. When stimulated with antigen, these T cells initiated cell division but failed to expand and showed poor production of the T cell effector molecules CD43, granzyme-B and IFN-γ. IL-7 and IL-21 did not rescue T cell expansion, although IL-21 enhanced CD43 and granzyme-B expression, but not IFN-γ production. Both IL-2 and IL-15 restored robust T cell expansion, which correlated with a strong up-regulation of Bcl-2 expression. Whilst IL-15 only restored CD43 and granzyme-B production, IL-2 rescued expression of all effector markers including the production of IFN-γ. Therefore, in terms of expansion and effector differentiation the IL-2-rescued T cells seemed equivalent to non-tolerant control T cells expressing the same MDM2-specific TCR. However, peptide titration experiments revealed that triggering of rescued T cells required higher peptide concentrations than triggering of non-tolerant control T cells. Together, the data show that while expansion and effector differentiation can be efficiently restored by cytokines, the impaired avidity of the rescued T cells might compromise their anti-tumor activity.
Materials and Methods
To generate pMDM100 specific TCR transgenic mice the Vα5 and Vβ7 TCR chain genes from a high avidity allo-MHC-restricted pMDM100-specific CTL clone [27], [28] were cloned into a CD2-vector (courtesy of Dr D. Kioussis) in preparation for microinjections into (C57BL/6×CBA) F1 (H2bxk) oocytes to produce transgenic mice. The transgenic mice were produced in collaboration with Dr M. Mendelson (Columbia University, USA). The Agneg mice (transgenic H2d mice) used in the experiments reported here were crossed back with BALB/c mice for at least five generations. The Agpos mice (transgenic H2dxb F1 mice) used were generated by mating the Agneg mice with C57BL/6 mice. Mice were typed for pMDM100-specific TCR expression by isolation of mouse-tail genomic DNA followed by polymerase chain reaction (PCR) screening. The oligonucleotide primer sequences used in the PCR reaction were: the CD2-5× forward primer (Invitrogen, UK): 5′-AACCCAGCTTTCCCTGAAAGTG-3′; the Vβ7-PSP reverse primer (Invitrogen): 5′-GCAGAATCCAGAATCAGGGAG-3′ or the Vα5-PSP reverse primer (Invitrogen): 5′-CACTGTCTCCCTCCCGGACAC-3′.
Peptides, Cytokines and Cell Lines
The synthetic peptide pMDM100 (ProImmune, UK) corresponds to amino acid 100-107 (YAMIYRNL) of the murine MDM2 protein. Synthetic peptide pSV9 (ProImmune, UK) corresponds to the amino acids (FAPGNYPAL) of the Sendai virus. In a previous study we have demonstrated that both pMDM100 and pSV-9 bind efficiently to H2-Kb MHC class I molecules [29]. Peptides were dissolved in PBS to give a concentration of 2mM and stored at −20°C. Human recombinant IL-2 (Roche), human recombinant IL-15 (R&D Systems), human recombinant IL-7 (R&D Systems) and murine recombinant IL-21 (R&D Systems) were added to selected T cell cultures. RMA-S cells (H2b) were derived from RMA cells by mutagenesis [30]. RMA-S cells are TAP deficient due to a point mutation in the TAP2 gene and express low levels of MHC class I molecules compared to RMA cells [29], [31]. MBL-2 cells (H2b) were derived from a Moloney murine leukaemia virus induced T-cell lymphoma [32].
Flow cytometric analysis
Spleen and lymph nodes (inguinal and lumbar) were harvested from 6–8 week old Agneg mice and Agpos mice and single cell suspensions obtained by macerating tissues through a 40 µm nylon cell strainer. Samples were stained in PBS (1% FCS) with the appropriate dilution of the relevant mAbs. Propidium iodide staining cells were excluded from analysis. Samples were acquired on an LSR2 flow cytometer (BD Biosciences, USA) and analysed using FACS Diva software. The following mAbs were used for flow cytometric staining: rat-anti-mouse Vβ7 FITC (Serotec, UK), rat-anti-mouse CD4 FITC, rat-anti-mouse CD8α APC, rat-anti-mouse CD8α Cy-Chrome 5 (CY-5), rat-anti-mouse Vβ7 PE, hamster-anti-mouse CD69 PE, rat-anti-mouse CD25 PE, rat-anti-mouse CD62L-FITC, rat-anti-mouse CD44 PE, rat-anti-mouse CD43 activation-associated glycoform-PE (all BD Biosciences).
To assess apoptosis, CD8+ T cells were isolated from splenocytes using a CD8α isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's protocol, stimulated as described, stained with annexin V and PI (both BD Biosciences) according to the manufacture's protocol and then analysed by flow cytometry as described previously.
For intracellular staining of Granzyme B and Bcl-2, T cells were permeabilized with BD cytofix/cytoperm kit (BD Biosciences) and stained with rat-anti-mouse Granzyme B PE mAb (eBioscience) or hamster-anti-mouse Bcl-2 FITC mAb (BD Biosciences). Specificity controls were performed using the appropriate isotype mAbs. Samples were analysed by flow cytometry as described previously.
Proliferation Assays
Antigen specific T cell proliferation was assessed by directly visualising the division of cells using the fluorescent cytosolic dye, Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE), or by the uptake of 3[H]-thymidine. For CSFE labelling of cells, splencoytes from the transgenic mice were incubated with 3 µM CFSE (Molecular Probes, UK) for 3 min at 37°C in 1ml of PBS. In each well of a 96 well-plate, 2×105 CFSE-labelled splenocytes were stimulated with 1×105 irradiated (80 Gy) temperature-induced RMA-S coated with pMDM100 peptide (10 µM) or an irrelevant control peptide, pSV9 (10 µM). The appropriate cultures were supplemented with 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. Cells were harvested at the appropriate time point, stained with CD8α and TCR Vβ7 mAbs and subjected to CFSE profiling by flow cytometry on CD8+Vβ7+ T cells. The proliferative response of the CTL in response to limiting concentrations of antigen was assessed in 3[H]-thymidine incorporation assays. Triplicate cultures were plated for each different experimental condition. 5×104 temperature induced irradiated (80 Gy) RMA-S cells were plated onto 96-well plates with wells containing graded concentrations of peptide in medium and were incubated for 1 hour at 37°C, prior to the addition of 2×105 transgenic splenocytes and 10 U/ml IL-2. Cells were incubated at 37°C in 5% CO2 for 2 days, pulsed with 0.5 µCi 3[H]-thymidine (Amersham, UK) and incubated for a further 24 hours. The cells were harvested using a 96-well plate harvester (Skatron Instruments, Norway) onto a filter mat and thymidine incorporation measured by liquid scintillation counting on a β-counter (Wallac, Finland).
IFN-γ assays
To measure antigen specific IFN-γ production, splenocytes from the transgenic mice were stimulated, culture supernatant harvested and IFN-γ measured in an IFN-γ ELISA. To compensate for the lower numbers of CD8+Vβ7+ T cells in the spleens of Agpos mice, IFN-γ production was assessed in cultures containing four-fold excess of splenocytes from Agpos mice compared to splenocytes from Agneg mice. Triplicate cultures were plated for each different experimental condition on 96-well round-bottom plates. In each well 1×104 splenocytes from Agneg mice or 4×104 splenocytes from Agpos mice were stimulated with 1×104 irradiated (80 Gy) temperature-induced RMA-S coated with pMDM100 peptide (10 µM) or an irrelevant control peptide, pSV9. In some experiments, peptides were added directly to the splenocytes of F1 mice. The appropriate cultures were supplemented with 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. For peptide titration the temperature induced irradiated RMA-S cells were plated onto 96-well plates with wells containing graded concentrations of peptide in medium and were incubated for 1 hour at 37°C prior to adding the transgenic splenocytes. After 72 hours, 50 µl culture supernatant was harvested from each well and murine IFN-γ was measured by sandwich ELISA using anti-IFN-γ antibodies (BD Biosciences). The activity in experimental samples was ascertained using the standard curve of mean absorbance values (OD) versus the dilution of recombinant IFN-γ in the supernatant.
The functional activity of IL-2 rescued CTL was measured by intracellular IFN-γ staining. Intracellular IFN-γ stainings were performed as previously described[28]. Samples were analysed using a FACScalibur™ flow cytometer and CELLQuest™ software.
IL-2 biosssays
To measure antigen specific IL-2 production, splenocytes from the transgenic mice were stimulated, culture supernatant harvested and IL-2 measured using IL-2 dependent CTLL cells. The stimulation phase of the IL-2 assay was performed exactly as the IFN-γ assay. After 72 hours, 50 µl culture supernatant was harvested, transferred to wells containing CTLL cells (5×103) and incubated for 16–18 hours. The cells were pulsed with 3[H]-thymidine by adding 1 µCi 3[H]-thymidine to each well and incubating for a further 12 hours. The activity in experimental samples was ascertained using the standard curve of cpm versus the dilution of recombinant IL-2 in the supernatant.
CTL assays
Cytotoxic activity was determined in 4-hour 51chromium-release assays against MBL-2 tumor cells and RMA-S cells coated with pMDM100 peptides or MHC class I-binding control peptides as described [33].
Transgenic T cells are present in the periphery of Agpos mice
Monoclonal antibodies against CD4, CD8 and Vβ7 were used to identify cells expressing the transgenic TCR β chain (antibodies against the transgenic TCR α chain were not available). Although thymic deletion occurred in Agpos mice (data not shown), a substantial number of T cells was found in the periphery. The staining profile of the lymph node samples demonstrated a significant percentage of CD8+ T cells that expressed Vβ7 in the Agpos mice (Figure 1 A), and a similar profile was observed for splenic T cells (data not shown). The mean fluorescence intensity (MFI) of Vβ7 staining for the T cells of Agpos mice was 3075 compared to 5543 in the Agneg control mice (Figure 1A). Similarly, there was a decrease in the CD8 expression in T cells of Agpos mice compared to in the Agneg mice (MFI of CD8 staining: 13907 and 32526). The decreased CD8 expression in the Agpos mice was seen only on the CD8+ T cells that expressed the transgenic Vβ7 TCR but not on those that did not. These data demonstrated that the MDM2-specific T cells in the periphery of Agpos mice had down-regulated CD8 co-receptor and TCR expression.
(A) Lymph nodes cells from 6–8 week old Agpos and Agneg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8+Vβ7+ cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.
Next the expression of the activation markers CD25, CD44, CD62L and CD69 was analysed on lymph node cells from Agpos and Agneg mice. The T cells from both sets of mice were CD25lo, CD69lo and CD62Lhi (Figure 1B). While the T cells of Agneg mice expressed intermediate levels of CD44, which is associated with a naïve phenotype, the T cells of the Agpos mice were CD44hi, which is characteristic of antigen experienced T cells (Figure 1 B).
T cells of Agpos mice display defects in antigen-driven expansion
We then analysed antigen-specific proliferation of the T cells in the Agpos mice and Agneg mice. Splenocytes from the mice were CFSE labelled, stimulated in vitro with pMDM100-coated RMA-S cells followed by staining with antibodies against CD8 and Vβ7 to identify transgenic T cells. The CFSE labelled T cells from the Agneg mice divided in response to pMDM100 stimulation, with each cell division producing an increase in the number of viable T cells containing reduced levels of CFSE (Figure 2A). Whilst the T cells from the Agpos mice divided a similar number of times in response to antigen-specific stimulation, we failed to see an accumulation of the divided T cells (Figure 2 A). CFSE labelled T cells from the Agneg or Agpos mice stimulated with a H2-Kb binding control peptide, pSV9, did not proliferate in these CFSE assays (Figure 2A).
(A) CFSE-labelled splenocytes from Agneg and Agpos mice were stimulated with RMA-S coated with pMDM100 peptide (10 µM) or pSV9 peptide (10 µM) for 48 hrs, stained with CD8 and Vβ7 mAbs and subjected to CFSE profiling by flow cytometry of gated CD8+Vβ7+ T cells. Data are representative of at least three independent experiments. (B) To measure antigen-specific IL-2 production, splenocytes from Agneg mice and Agpos mice were stimulated with RMA-S cells coated with pMDM100 peptide (100 µM) or a control peptide, pSV9 (100 µM). After 72 hours, 50 µl of culture supernatant was harvested and murine IL-2 was measured in a CTLL bioassay. Data represent the mean±SD of triplicate values and are representative of two independent experiments. (C) CFSE-labelled splenocytes from Agneg mice were stimulated with RMA-S cells coated with pMDM100 or SV9 control peptides for 72 hrs. In the bottom panel, CFSE-labelled T cells were stimulated for 72h with pMDM100 in the presence of unlabelled splenocytes from Agpos mice. (D) CFSE-labelled splenocytes from Agpos mice were stimulated with RMA-S cells coated with pMDM100 or SV9 control peptides for 72 hrs. In the bottom panel, CFSE-labelled T cells were stimulated for 72h with pMDM100 in the presence of unlabelled splenocytes from Agneg mice.
As IL-2 is a major autocrine growth factor produced by functionally competent CD8+ T cells, we explored if T cells of Agpos mice were able to express this cytokine. While non-tolerant control T cells of Agneg mice secreted IL-2 upon peptide stimulation, no IL-2 was detectable when the T cells of Agpos mice were stimulated (Figure 2B). Together, this suggested that the T cells displayed an anergic phenotype characterised by a lack of IL-2 production and an inability to expand upon peptide stimulation. Thus, we sought to determine whether the anergic T cells displayed regulatory activity and inhibited the proliferation of functionally competent T cells. To do this, CFSE labelled T cells from the Agneg mice were co-cultured with unlabelled tolerant T cells and stimulated with pMDM100 for 72 hours. CFSE-labelled non-tolerant T cells showed peptide-specific proliferation regardless of whether or not tolerant T cells were present in the cultures (Figure 2C). As expected, CFSE-labelled tolerant T cells showed defective expansion when stimulated with pMDM100 peptides. This defect was reversed when tolerant T cells were stimulated with peptide in the presence of non-tolerant T cells (Figure 2D), indicating that functionally competent T cells were able to restore the expansion of tolerant T cells. As non-tolerant T cells can produce IL-2 upon peptide stimulation (see above) it is possible that this cytokine may have triggered the expansion of the peptide-stimulated tolerant T cells. To explore this further, we tested directly whether IL-2 or other common gamma chain cytokines were able to restore the expansion of the tolerant T cells.
IL-2 and IL-15 efficiently induce Bcl-2 expression and restore the expansion of tolerant T cells
CFSE-labelled T cells of Agpos mice were stimulated with peptide antigen in the presence of IL-2, IL-7, IL-15 or IL-21. FACS analysis revealed that peptide stimulation in the presence of IL-2 and IL-15 resulted in robust expansion of tolerant T cells (Figure 3A). The observed kinetics of cell division and cell accumulation was similar to that seen with peptide-stimulated non-tolerant control T cells of Agneg mice. In contrast, tolerant T cells stimulated with peptide and IL-7 or IL-21 showed a defect in the accumulation of divided cells similar to that seen after peptide stimulation without cytokines (Figure 3A). This indicated that IL-7 and IL-21 were unable to rescue the defective expansion of tolerant T cells.
(A) CFSE-labelled splenocytes from Agneg and Agpos mice stimulated with RMA-S coated with pMDM100 peptide (10 µM) for 72 hrs in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21, stained with CD8 and Vβ7 mAbs and subjected to CFSE profiling of gated CD8+Vβ7+ T cells. (B) Splenocytes from Agneg mice and Agpos mice were stimulated for 3 days in vitro with pMDM100-coated targets in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21 and Bcl-2 expression levels were analysed by intracellular staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8+Vβ7+ population. Following staining of the splenocytes with an appropriate isotype control mAb the MFI of the gated CD8+Vβ7+ population from the Agneg mice and Agpos mice was 98 and 97, respectively. Data in A and B are representative of three independent experiments. (C) CD8+ T cells isolated from the spleens of Agneg and Agpos mice were stimulated with RMA-S coated with pMDM100 peptide (10 µM) in the presence or absence of 10 U/ml IL-2 and cell death was determined on the indicated days by staining with annexin-V and propidium iodide and flow cytometric analysis.
Next, we explored whether IL-2, IL-7, IL-15 or IL-21 enhanced the expression of the anti-apoptotic molecule Bcl-2 in tolerant T cells. Peptide stimulation in the absence of cytokines revealed that the tolerant T cells expressed substantially less Bcl-2 than the non-tolerant control T cells (Figure 3B). However, stimulation in the presence of IL-15 and IL-2 resulted in higher levels of Bcl-2 expression in tolerant T cells compared to the control T cells. Although IL-7 and IL-21 were able to up-regulate Bcl-2 expression in tolerant T cells, the levels were lower than those seen after IL-2 and IL-15 stimulation. These data showed that the efficient Bcl-2 up-regulation seen with IL-2 and IL-15 correlated with the ability of these cytokines to rescue the expansion of tolerant T cells. Further experiments revealed that the defective expansion of tolerant T cells correlated with a high rate of apoptosis as revealed by annexin-V and propidium iodide staining (Figure 3C). When tolerant T cells were stimulated in the presence of IL-2 they showed a low level of apoptosis similar to that seen in non-tolerant control T cells. Thus, it is possible that up-regulation of Bcl-2 resulted in the suppression of apoptosis which promoted the expansion of the tolerant T cells. Alternatively, it is possible that IL-2 and IL-15 activated additional pathways required for T cell expansion, and that these pathways were not activated by IL-7 and IL-21.
Rescue of defective effector differentiation of tolerant T cells
We then analysed the effect of the common gamma chain cytokines on the differentiation of tolerant lymphocytes into effector T cells. We measured IFN-γ secretion as this effector molecule is typically produced by CD8+ T cells. We also measured expression of the serine protease granzyme-B as it plays a key role in the granule-exocytosis pathway of CTL mediated target cell killing [34], [35]. Finally, we analysed the expression of the activation-associated glycoform of CD43 which has been shown to be up-regulated on effector T cells [36].
Upon peptide stimulation in the absence of cytokines, the tolerant T cells expressed lower levels of CD43 than non-tolerant control T cells. Stimulation of tolerant T cells in the presence of IL-7 resulted in a modest CD43 up-regulation. Both IL-15 and IL-21 triggered more substantial CD43 expression than IL-7, although the highest CD43 levels were induced by IL-2 (Figure 4A). Interestingly, IL-2 had only a modest effect on CD43 expression in non-tolerant control T cells, where IL-21 mediated the strongest CD43 up-regulation (Figure 4A).
Splenocytes from Agneg mice and Agpos mice were stimulated for 3 days in vitro with pMDM100-coated targets in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. (A) CD43 expression levels on the stimulated T cells was analysed by surface staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8+Vβ7+ population. (B) Granzyme B expression levels on the stimulated T cells was analysed by intracellular staining and flow cytometric analysis. Numbers in histograms represent the specific MFI of the gated CD8+Vβ7+ population. Following staining with an appropriate isotype control mAb the MFI of the gated CD8+Vβ7+ T cells from the Agneg mice and Agpos mice was 398 and 495, respectively. Data in A and B are representative of three independent experiments. (C) Cytolytic activity of stimulated T cells against MDM2-expressing MBL-2 tumor cells and RMA-S targets coated with pMDM100 peptide (10 µM) or a class I binding control peptide, pSV9 (10 µM). Data are representative of two independent experiments.
The granzyme-B expression pattern of tolerant T cells was similar to that of CD43. Peptide stimulation in the absence of cytokines resulted in reduced granzyme-B expression in tolerant T cells compared to non-tolerant control T cells (Figure 4B). IL-2 was most effective in up-regulating granzyme-B expression, followed closely by IL-21 and IL-15, while IL-7 had only a modest effect. Finally, the granzyme-B expression pattern correlated with the cytotoxic activity of tolerant T cells. T cells rescued by IL-2, IL-15 and IL-21 killed peptide coated target cells and tumor cells more efficiently than T cells cultured with IL-7 or without cytokine supplementation (Figure 4C).
The analysis of the effector molecule IFN-γ revealed that tolerant T cells were unable to secrete IFN-γ after peptide stimulation in the absence of cytokine, whereas non-tolerant control T cells readily produced IFN-γ (Figure 5A). Whilst IL-2 efficiently restored IFN-γ secretion by tolerant T cells, the other common gamma cytokines IL-7, IL-15 and IL-21 were unable to promote IFN-γ production (Figure 5B).
(A) To measure antigen-specific IFN-γ production, splenocytes from Agpos mice and Agneg mice were stimulated with RMA-S cells coated with pMDM100 peptide (10 µM) or a control peptide, pSV9 (10 µM). After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent the mean±SD of triplicate values. (B) Antigen-specific IFN-γ production by splenocytes from Agpos mice stimulated with pMDM100 peptide or a control pSV9 peptide in the presence of 10 U/ml IL-2, 10 ng/ml IL-7, 50 ng/ml IL-15 or 50 ng/ml IL-21. After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent the mean±SD of triplicate values. Data in A and B are representative of three independent experiments. (C) Antigen-specific IFN-γ production upon secondary peptide stimulation of IL-2 rescued T cells was measured by intracellular staining for IFN-γ. T cells were re-stimulated with RMA-S cells coated with pMDM100 (10 µM) or an irrelevant control peptide, pSV9 (10 µM), for 5 hours in the presence or absence of 10 U/ml IL-2, followed by intracellular staining for IFN-γ. Comparable results were obtained when IFN-γ production by IL-2 rescued T cells was measured by ELISA in two independent experiments.
Taken together, IL-2 was most effective in restoring all analysed defects of effector differentiation of tolerant T cells. The activity of IL-2 was similar to that of IL-15 and IL-21, except that the latter two cytokines only rescued CD43 and granzyme-B expression but were unable to restore IFN-γ production by tolerant T cells.
Subsequently we tested whether cytokine rescued T cells became functionally equivalent to non-tolerant T cells, or whether they remained cytokine dependent upon secondary peptide stimulation. Thus, tolerant T cells were first stimulated with peptide and IL-2 for 7 days, washed and re-stimulated with peptide in the absence or presence of IL-2. Secondary stimulation in the absence of cytokines resulted in poor IFN-γ production by tolerant T cells (5% positive T cells versus 2% background with the control peptide), whereas re-stimulation in the presence of IL-2 restored IFN-γ production by 33% of the T cells (Figure 5C). As expected, the secondary IFN-γ response of non-tolerant control T cells did not require the presence of IL-2. Peptide stimulation in the absence of IL-2 supplementation resulted in IFN-γ production by 23% of T cells, which increased to approximately 42% in the presence of IL-2. Together, these experiments indicated that IL-2 rescued tolerant T cells remained IL-2-dependent when re-challenged with peptide antigen.
The cytokine rescued tolerant T cells display reduced functional avidity
As IL-2 rescued tolerant T cells seemed functionally equivalent to the non-tolerant control T cells we assessed the avidity of these two T cell populations. IL-2 rescued T cells and control T cells were stimulated with decreasing concentrations of peptide followed by measurement of IFN-γ production. IL-2 rescued T cells required higher peptide concentrations to trigger IFN-γ production than non-tolerant control T cells (Figure 6A). Similarly, antigen-specific proliferation required higher peptide concentrations when IL-2 rescued tolerant T cells were compared with non-tolerant control T cells (Figure 6B). Therefore, despite expressing the same transgenic TCR, the cytokine rescued T cells were of lower functional avidity than the non-tolerant control T cells.
(A) Splenocytes from Agneg mice and Agpos mice were stimulated with RMA-S cells coated with titrated concentrations of pMDM100 peptide or an irrelevant control peptide, pSV9, in the presence of 10 U/ml IL-2. After 72 hours, 50 µl of culture supernatant was harvested and IFN-γ was measured by ELISA. Data represent mean±SD of triplicate values. IFN-γ produced in response to RMA-S loaded with pSV9 was subtracted from all values. Data are representative of two independent experiments. (B) To measure antigen specific proliferation, splenocytes from Agneg mice (black squares) and Agpos mice (grey circles) were stimulated with RMA-S cells coated with titrated concentrations of pMDM100 peptide or an irrelevant control peptide, pSV9, in the presence of 10 U/ml IL-2 for 2 days, and then pulsed with 0.5 µCi 3[H]-thymidine for 24 hours. The maximal proliferative response by splenocytes from the Agneg and Agpos mice was 130480.2±9552.9 CPM and 56225.17±2794.9 CPM (triplicate values), respectively.
In this study we defined how physiological expression of the endogenous MDM2 protein shapes the response profile of T cells expressing an MDM2-specific TCR. The peripheral T cells responded to MDM2 peptides by initiating cell division, but the cells were unable to expand which correlated with a block in IL-2 production. This resembled the phenotype of the CD8+ T cells from protein kinase C-θ (PKCθ)-deficient mice [37]. CD8+ T cells in PKCθ-deficient mice underwent antigen-induced proliferation but displayed impaired survival and therefore failed to accumulate. Exogenous IL-2 or culture with wild-type T cells could correct the defective expansion of the PKCθ-deficient T cells, which is similar to the observations with the tolerant T cells analysed in this study. Therefore, it is tempting to speculate that a defect in the PKCθ pathway may underlie the defective expansion of the MDM2-specific T cells from Agpos mice.
We found that IL-2 was able to rescue the expansion and effector differentiation, including IFN-γ production, of tolerant T cells. Surprisingly, IL-15 was unable to rescue IFN-γ production by the tolerant MDM2-specific T cells. As previous studies have demonstrated that IL-15 stimulation can promote IFN-γ production by murine and human T cells [38], [39], this pathway seems to be defective in the tolerant T cells studied here. The observation that IL-15 stimulation enhanced T cell expansion and expression of certain effector markers indicated that the tolerant T cells were responsive to IL-15, but retained a selective defect in the pathway leading to IFN-γ expression. Furthermore, the differential effects of IL-2 and IL-15 in tolerant T cells indicated that these two common gamma chain cytokines use distinct mechanisms to trigger the production of IFN-γ. Whilst previous studies have identified a transcriptional control mechanism by which IL-2 induced IFNγ expression [40], the mechanism of IL-15-mediated IFN-γ production is currently less clear.
IL-21 was unable to rescue the expansion of the tolerant T cells, but was able to up-regulate the expression of CD43 and granzyme-B. Like IL-15, IL-21 did not promote IFN-γ production of peptide-stimulated tolerant T cells. Recent studies have shown that IL-21 can work synergistically with IL-15 to promote expansion and effector differentiation of naïve T cells in mice and in humans [38], [41]. It will be interesting to study how combinations of common gamma chain cytokines affect the function of the MDM2-specific T cells. A major advantage of IL-15 and IL-21 is that they can maintain the expression of the T cell co-stimulatory molecule CD28 and lymphoid homing markers such as CD62L [38], which was found to improved the in vivo survival and anti-tumor activity of adoptively transferred T cells in murine model experiments [39], [41], [42].
Recent studies analysed T cell tolerance of transgenic T cells expressing TCRs specific for the murine homologue of the melanoma antigen gp100 and the gag protein of FMuLV [16], [17]. The gp100-specific T cells seemed little affected by antigen expression in normal melanocytes, as they were functionally competent when stimulated with peptides ex vivo [16]. In contrast, the peptide stimulation of the gag-specific T cells showed that they were unable to proliferate, although they developed cytotoxicity and produced IFN-γ. IL-2 was relatively inefficient in reversing the proliferative defect of the gag-specific CTL, while IL-15 was highly effective in this model [17], [43]. The analysis of MDM2-specific CTL showed that both IL-2 and IL-15 restored T cell proliferation and expansion, indicating differences in the mechanism of tolerance induced by gag and MDM2. It is likely that the tissue-specificity and level of antigen expression determined the level of tolerance in the three transgenic models. The gp100 expression in melanocytes had little effect on T cell function, MDM2 expression in most normal tissues induced tolerance that was readily reversed by IL-2, whilst the expression of gag as a transgene using the albumin promoter rendered tolerant T cells relatively unresponsive to IL-2. Together, these TCR transgenic models provide valuable information about how distinct expression patterns in normal tissues affects the state of tolerance of TAA-specific T cells. Furthermore, these models can be used to gain insights into possible strategies to reverse tolerance of TAA-specific CTL.
We found that IL-2 was effective in restoring the function of MDM2-specific T cells ex vivo, although this did not lead to a complete restoration of T cell avidity. A possible explanation for the lower avidity of rescued T cells is the reduced expression levels of the TCR and the CD8 co-receptor seen in the tolerant T cells. It is interesting to note that MDM2 presentation in Agpos mice was sufficient to trigger TCR down-regulation, although previous studies suggested that effector CTL did not recognise normal tissues. For example, high avidity effector CTL specific for the same MDM2 peptide analysed here, selectively killed MDM2 expressing tumors but not normal cells [27], [28]. These observations suggested, that the antigen threshold required for the induction of tolerance in the transgenic T cells was lower that the threshold required for the triggering of cytotoxic effector CTL. However, it is possible that effector CTL remain susceptible to the tolerogenic signals that diminish the function of the tolerant T cells. We observed that adoptive transfer of MDM2-specific effector CTL into tumor-bearing resulted in the induction of T cell unresponsiveness [28]. This unresponsiveness was not due to tumor induced immune suppression, but was also seen after CTL transfer into tumor free animals. This is compatible with the idea that antigen expression in normal tissues can induce tolerance in adoptively transferred CTL, even when antigen levels are insufficient to trigger effector function. Thus, it is likely that adoptive T cell therapy needs to be combined with strategies that can protect transferred T cells from the induction of unresponsiveness. One of the beneficial effects of IL-2, which is typically administered in combination with adoptive T cell transfer [16], [43], [44], may relate to its ability to prevent anergy induction in transferred T cells.
Author Contributions
Conceived and designed the experiments: HS GB SX AH. Performed the experiments: GB SX AH. Analyzed the data: HS GB SX AH. Wrote the paper: HS GB.
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text:
F-22 Data Compression
Award Information
Small Business Information
3915 Germany Lane, Suite 102, Beavercreek, OH, 45431
Principal Investigator:
James Utt
Principal Investigator
(937) 431-1644
Principal Investigator
(937) 431-1644
Business Contact:
Ray Trimmer
Executive Vice President
(937) 431-1644
Executive Vice President
(937) 431-1644
Research Institution:
AbstractThe F-22 Data Compression program addresses the problem of the insufficient imagery storage capacity of the F-22. This impacts the ability of the aircraft to deploy munitions or transfer imagery for tactical planning. The imagery capacity of the targeting system is significantly smaller than the area of operations thus restricting the ability to attack targets. For reconnaissance operations, due to the large size of the images, the amount of data that can be recorded is limited and must be manually processed and distributed. The data compression algorithms used by the DRA/UtopiaCompression team will allow the F-22 SAR imagery to be compressed using a pattern driven approach whereby images and, specifically, regions in images with defined visual characteristics are considered as relational entities, classifiable according to the presence or absence of patterns, their relationships, and the frequency of their occurrence. The algorithm seeks to minimize the set of extracted features with the application of statistical machine learning to extract features and model patterns. The codec is trained on such patterns to "learn" their nature so that variable-sized segments of the image can be accurately reconstructed. The algorithm results in a five to ten fold improvement over JPEG compression technology.
* information listed above is at the time of submission.
An Official Website of the United States Government

subset: commoncrawl


text:
Galway Festivals & Events
Galway City and surrounding county plays host to a wide range of festivals and events throughout the year and have something for all ages and interests. The Claregalway Hotel is the perfect choice for accommodation when attending any of the many events and festivals.
Galway Races - Summer Festival Meeting
Adrenalin-pumping action at Galway racecourse, Ballybrit from July 25th to July 31st, 2016.
Clarenbridge Oyster Festival
A celebration of the native Oyster, the history of this beautiful village and its traditions. 9th–11th September 2016, Clarenbridge
Larry Reynolds Weekend
Traditional Irish music, culture and heritage, promoting the East Galway Style of Music. 9th – 11th September 2016, Ballinasloe
Galway Tattoo Show
International tattoo show, best tattoo artists. Entertainment, live music, art gallery, market stalls&more. 10th–11th September
Shorelines Arts Festival
Celebrate art and sculpture from local artists with guests. Vintage machinery display, art talks&more. 15th-18th Sept, Portumna
Clifden Arts Festival
Lots of events and live music performances. 15th – 25th September, Clifden.
Galway International Oyster Festival
The picturesque town of Galway hosts the Galway International Oyster Festival from September 23rd to 25th, 2016.
National Hot Air Balloon Championships
40 competing teams from all over the world such as USA, England, Scotland, Wales, Spain, Italy and more. 25th-30th September, Loughrea.
Burren Peaks Walking Festival
Guided hill and lowland walks for all walkers. Enjoy limestone pavement-Karst landscape. 23rd–25th September 2016
Baboró International Arts Festival for Children
Takes place in Galway City from October 17th to October 23rd, 2016. Fun for all the family.
Vodafone Comedy Carnival Galway
Formerly known as The Galway Comedy Festival. Takes place in October 2016. Stay tuned for more details.


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A dry start to spring has caused water levels in dams which supply water to towns across Western Australia's south-west and Great Southern to fall to nearly a third of capacity.
Topics: dams-and-reservoirs, water-supply, water, collie-6225, albany-6330
WA Government-owned electricity generator Synergy reaches an agreement to sell surplus coal to the operators of a South West alumina refinery plagued by supply issues.
Topics: coal, mining-industry, state-parliament, bunbury-6230, collie-6225
A 54-year-old man has been killed in a traffic crash near the south-west Western Australian town of Collie.
Topics: road, collie-6225, bunbury-6230
Posted| Updated
A man who jumped into a river to a save an elderly driver and his pet dog from a sinking car is one of two West Australians recognised in the Australian Bravery Awards.
Topics: people, emergency-incidents, molloy-island-6290, collie-6225
A Collie woman has pleaded guilty to stealing $444,278 from exploration company Musgrave Minerals while she worked for the company.
Topics: burglary, fraud-and-corporate-crime, collie-6225
Detectives have charged a 30-year-old Collie man over an armed hold-up at a Bunbury pharmacy in south-west Western Australia.
Topics: armed-robbery, bunbury-6230, collie-6225
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text:
Ian Hurst - arrives to give evidence at The Leveson Inquiry at The Royal Courts of Justice on November 28, 2011 in London, England. 21 victims of phone hacking will be giving evidence at the inquiry over the next five days. The inquiry is bei - Monday 28th November 2011 (7 images)
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Looks fruity to me, I'll say apple.
looks like elm to me
Guy at work just bought 5 cord green delivered for 1200. Seller had quoted 275.00 per cord but gave him a discount to make the deal.But...
Been using a clean burn hot air furnace firing on waste automotive oils to heat a garage for a couple of winters now and am very pleased. 1200...
Heating with a defiance volcano here and have the output ducted to a floor register in the kitchen directly below the stove, been that way for...
Wow! Amazing craftsmanship. If this ends up working as good as it looks I may be interested in purchasing the next one you make, what will it cost me?
Looks like a silver maple but more pics would help. Musta made a pretty good thump when it landed.
With that much wood to cut for free it would justify upgrading the saw. You may be surprised at how much time it will save bucking rounds. I ran...
Are you sure its Hard maple?
Another soft maple vote here.
I say, keep in mind that all the firewood to feed that stove has to come into the main living area and can be messy. My wife "wood" not like wood...
Seasons fast burns quick with little to no coals. Great for shoulder season.
I say both are soft maple.
Your still young and wood burning is probably in your blood. Next thing you know you'll be running low on wood and fretting about finding more....
I also say white oak nice stuff!
I say poplar also, knee jerk was hickory but look at those wide growth rings!
Looks like popular to me. Very light when dry, easy to split, burns quickly leaving no coals. I've burned lots of it, best used during shoulder...
I'm also in southern nh and would like to get contact info for this supplier if possible Thank you.
sure looks like silver maple to me and I'll guess 3/4 cord
It doesn't appear to be a fisher. From what I can make out from the lettering on the door it is a warm morning model.
Only thing missing is the benny hill music in the background.
Split does not look like oak to me either. Maybe popple? Rear window on the P/U looks drafty!
Check out harbor freight they have chinese engines and are quite reasonably priced. Just bought a 5.5 horizontal for 99.95 w/coupon for my...
Looks like ya got some box elder!
Ya, looks like a mess to clean up but that there's some primo kindlin' esp. the quills
Hmmmm...The smell of gas at the end of part I has me wondering....
looks like red maple to me
Looks like soft maple, I'll say silver
Given the amount of fungus growing out of it, I am not all that suprised that it's heat value is low.


text: Well tomorrows the day! Lol, the day I see if my period really is going to happen or not. I didn't have a period FORVER not last month but the month before. I had a forever period and a forever not period until my husband came home, then I was normal again. That speaks to how stressful it was to have him gone for 2 months! Makes me wonder if it was more than stress that contributed. What if I don't have a period for forever again? I don't WANNA go to the doctors until Im pregnant! I always chicken out! So Im hoping for either my period or a positive pregnancy test. If not, next month me and my hubby will actually try to get pregnant when Im fertile. Again, Im worried about working around chlorine and maybe being pregnant and that causing a mis carriage or lack of fertility or just bad health for a developing baby. It's gunna be hard to quit tho, I really love working there! It's seriously the best working environment I've ever been at! It's nice to know my parents are getting excited now, they weren't really before. Im scared about all this, but also excited and happy. I think I need to just do it, cuz if I don't I'll just want to really bad, and if I do it, it'll be different but rewarding. Chickening out is lame sauce.


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Staff & Faculty
You are here: Home » Campuses » TSiBA Eden » Staff & Faculty
Sam Noemdoe
Sam is the Eden Branch Manager overseeing operations, delivery and rural strategy. Sam holds a Science Degree and B.Ed, an Honours in Business Administration and MBA.Teaching is his core profession and having spent significant time in corporate, in the employee wellness industry, and running his own businesses he developed his entrepreneurial skills.
Alex Zukisile Xokozela
Alex manages the academic and theoretical components of our Practical Certificate programme. Alex has taught accounting, and guides our students in the theories and ethics underlying business and entrepreneurship. Alex has a National Higher Certificate, majoring in Accounting and Financial Management, and is a Registered Assessor with the Services Seta.
Genevieve Keene
Genevieve is Eden's Sustainability Manager holding strategic partnerships for funding and the TSiBA Education Solutions offering. Genevieve has a Masters of Social Sciences Degree (Counselling Psychology), passion for businesses and rural community development.
Bongani Masuku
Bongani has recently joined our Eden Team as our CPBA (Certificate in Business Administration) Practical Programme Supervisor.
Adrian Van Rooyen
Adrian lectures in Math and IT. He holds a B.Com degree with majors in Accouting and Auditing. Adrian has three years experience working at an Auditing and Accounting firm in George, and he is currently completeing his Honours degree in Accounting and Magagement Accounting.
Richard Sedi
Richard Sedi is the Operations Coordinator and Bookkeeper. He is responsible for bookkeeping, operations and stock and assets management. Richard has a N6 Diploma in Financial Management.
Francine Rubin
Francine Rubin is the Environmental Coordinator. Her role involves teaching Environmental Education and providing guidance to the Organic Vegetable Student Business and the Campus Green Audit committee. She holds a Master's in Environmental Management.
Karon Barnard
Karon is the Academic Manager at Eden Campus and responsible for the ETQA of programme delivery and the application of the QMS throughout the campus. She has experience with SETA accredited and DHET programmes. She holds a BTech degree (Hons) specialising in Adult Education and Nat. Dip. Business Management.
Willem Olivier
Willem Olivier is in charge of carrying out campus maintenance according to the year plan.
Bridgette Hasley
Bridgette Hasley is the Kitchen Manager. She places food orders, manages stock and and keeps the kitchen budget records up to date.
Dee Francis
Dee is the LSD-F Co-ordinator and she is in charge of Student Support on campus.
Anelene Kees
Anelene is the Office Administrator. She keeps the campus moving smoothly.
Vukile Witbooi
Vukile is a Higher Certificate Business Administration (HCBA) Lecturer for TSiBA Eden students.
Marilyn Meyer
Marilyn is the Ignition Centre Administrator and is responsible for Career Guidance, Basic Computer Skills Training, Alumni Tracking & Community Training. Marilyn has a TSiBA CPBA and HCBA and experience in facilitation & entrepreneurial support.
Stephen Brown
Stephen is the Hostel Co-ordinator and Driver.
Angelique Jantjies
Angelique Jantjies is the Cooking Co-ordinator.
Fernandez Pedro
Fernandez Pedro is the Ignition Centre Coordinator at the Eden Campus responsible for the delivery of the TSiBA Education Solution's offerings, recruitment, entrepreneurial & employment support as well as marketing. Fernandez holds a degree in mathematics & computer science and an honors degree in business management.


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We won't see another college basketball game for about seven months. That's a long time.
But the players in the highly touted 2013 recruiting class will reach their respective campuses soon.
Future Arizona forward Aaron Gordon (No. 4 in the 2013 class per RecruitingNation) could be a game-changer who morphs Sean Miller's squad into a Final Four-caliber program. He's Blake Griffin Lite.
Indiana's Noah Vonleh (No. 8), a 6-foot-8 five-star forward, could make folks in Bloomington forget about the loss of Victor Oladipo and Cody Zeller.
Former Nerlens Noel teammate Wayne Selden (No. 12) is a stocky 6-foot-5 guard with swagger. He's going to Kansas and, yes, he'll log minutes for Bill Self.
Florida's Billy Donovan will have a good rotation problem when McDonald's All Americans Kasey Hill (No. 9) and Chris Walker (No. 14) enter the mix in Gainesville.
Locals are at the center of Memphis' nationally ranked recruiting class, which includes PF Austin Nichols (No. 15), SF Kuran Iverson (No. 31) and SF Nick King (No. 33).
Duke's Jabari Parker (No. 2) plays like a pro. He'll be ready to star in Mike Krzyzewski's system in Year 1.
And then there's the young NBA franchise that John Calipari signed. The "greatest recruiting class of all time" is a legitimate label for the top-rated bunch in the field. Julius Randle (No. 3), Andrew Harrison (No. 5), James Young (No. 6), Aaron Harrison (No. 7) and Dakari Johnson (No. 11) are all potential pros.
Here's what some of the coaches who'll have to deal with those young studs in conference play next season had to say (anonymously) about the impact they might have on their programs as freshmen.
Aaron Gordon (Arizona)
"Arizona will be very tough adding Gordon. He's versatile and athletic and could probably play three to four positions." -- Pac-12 head coach.
Noah Vonleh (Indiana)
"Noah is as talented as any 2013 recruit. He has the total package. Should be an impact player from day one. He's very skilled for his size and also has a college-ready body." –-- Big Ten assistant.
Wayne Selden (Kansas)
"He's a big-time athlete and very talented, I know that." -- Big 12 head coach.
Kasey Hill and Chris Walker (Florida)
"Hill is a special talent and will play, but he is not beating out [Scottie Wilbekin]. He is too tough and the best defender in our league. I worry about Walker. Will Walker accept his role? Because he is not a better basketball player than [Dorian Finney-Smith] or [Damontre Harris]. He is a better athletic talent but not a better basketball player." -- SEC head coach.
Austin Nichols, Kuran Iverson and Nick King (Memphis)
"I think from what I saw, Kuran Iverson has the intangibles to make an impact. Nick King has good length and size. He has the best chance to make an impact. Austin Nichols is going to need a year to develop his body." -- American Athletic Conference/Big East assistant.
Jabari Parker (Duke)
"Jabari has the ability to impact the game in so many ways. He's versatile, knows how to win, and will fit in very well in Duke's style." -- ACC assistant.
Aaron Harrison, Andrew Harrison, Julius Randle and Dakari Johnson (Kentucky)
"I think they're all very talented. When you're talking about this class, they all have the mentality of one-and-done. Now you have to make it work. Two years ago, they had some experienced guys with a seven-man rotation. Now, they'll have a nine- or 10-man rotation." -- SEC head coach.


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Nine wrestlers from Southern Regional High School and one from Pinelands Regional are still alive for Region 6 titles on Friday at Pine Belt Arena in Toms River.
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The old saying that there is no such thing as a free lunch didn't stop a number of New Jersey school employees from trying. A troubling recent report by the state comptroller uncovered widespread fraud in lunch programs that the federal government subsidizes to feed needy children.
The investigation identified more than 100 people who allegedly lied about their income so that their children could get free or reduced-price meals. Even more egregiously, the culprits or their family members were on public payrolls, and they underreported their incomes by a staggering $13 million to get discounted meals. Among those caught milking the system were 40 school district employees and six school board members in Pleasantville, Newark, and Paterson.


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Parents & Family
Rowan hosts Attracting Women into Engineering for middle school girls | More
"My Little Hundred Million": Best-selling author's podcast celebrates Henry Rowan, explores higher ed philanthropy | More
Rowan student journalists to cover Democratic National Convention | More
Rowan's Online Construction Management degree cited as a "best value" | More
Rowan Engineering welcomes BEST young men | More
Whether they admit to it or not, your kids love to hear from you. There is no better way to say Happy Birthday, Just Thinking About You, or I Miss You than an e-card. They are fun for you (picking out the right one), and they are fun for your student to recieve.


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As part of the BUS 450 International Business course in Term 7, instructor John McGuire arranged for the 18 MAP students enrolled in the class to visit the Islamic School of Indianapolis. Students were welcomed by the school principal, Mr. Tewfik Choukri, who is originally from Morocco, and Islamic art teacher, Mr. Najib El-Gouche, born in Lebanon.
Choukri and El-Gouche freely discussed the Islamic culture and answered students' questions on topics such as treatment of women, terrorism, and Sharia business practices. While there, students were allowed to quietly observe the evening call to prayer at sunset, one of five times each day that Muslims traditionally pray. "The event was provocative for thought and an enjoyable experience on a subject that is consistently in world news," said McGuire.
MU-COM New Student Orientation
Aug 01
Early Move-In: Football Team
Aug 02
White Coat Ceremony
Aug 07
Early Move-In: House and Apartment Residents, Marching Band...
Aug 08
2016 USA Cycling Masters Track National Championships
Indianapolis |
Aug 09


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Managing email rules
Managing email rules
Managing e-mail rules in Outlook means editing rules that already exist, deleting rules, and most importantly, sequencing the rules that you've created. To manage our rules, we'll choose Rules on the Home tab of the Ribbon and choose Manage Rules & Alerts. This opens the Rules and Alerts dialog box. Here, you see the four rules that we've created previously in this title: OilFest 2010 and Move Vendor Info Requests, which we've created in the last video, and the rules that move messages from Arthur Lot and messages from LinkedIn, which we created earlier.
These rules will be applied, as you note, in the order shown. So, OilFest 2010, when a message comes in, Outlook will grab and say does it meet the criteria listed in OilFest 2010? If so, it will take action. If not, then it moves down the list, and it says, well, does this meet the criteria listed in the rule called Move Vendor Info Requests? If so, I have some work to do, and it not, it goes on to see if it's from...
Managing email rules provides you with in-depth training on Business. Taught by Gini Courter as part of the Outlook 2010: Effective Email Management
- Subject:
- Business
- Software:
- Outlook
- Author:
- Gini Courter
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APHA-PtHA AQHA -Palomino and Buckskin. Training, Showing, Lessons, Fitting , Sales and Evaluations
facility includes 100x 200 outdoor arena, 60 ft round pen, 4-horse hotwalker, 10 stall barn, 6- 50x150 turn-outs with run in shelters, 3 large turn…
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Cutting and Reined Cow Horse Trainer
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We have full and partial horse boarding 12x12 matted stalls,Eastern Ore Orchard hay fed 2 Hay for lunch Stalls cleaned daily,daily turnout,We are…


text: Santorum: Yes, That Is My Shirtless PictureMaureen O'Connor3/16/12 7:05pmFiled to: 2012 electionsRick SantorumnipplesshirtlessNip SlipTweetgRoundup126EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Like Barack Obama and Mitt Romney before him, GOP presidential candidate Rick Santorum has been photographed naked from the waist up. Blogger Joe My God has uncovered an image of Santorum sunbathing poolside in Puerto Rico. Now that we have finally seen every viable presidential candidate's nipples, I'm ready to call this one for Obama. I'm not saying Romney's invisinipples make him unfit for public office, just that areolae-conservative voters may not be ready for so little eumelanin. Advertisement In a radio interview today, Santorum confirmed the photograph was real. "They caught me sunbathing," he acnowledged. Joe My God writes that a passenger from an all-guy cruise—which happened to stay at the resort at the same time as Santorum—snapped the photo. [Joe My God, Buzzfeed, Buzzfeed]Reply126 repliesLeave a reply


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National Esthetic Teacher Training Conference 2016
Registration is Open
When: Saturday, August 20, 2016
9:00 am - 5:45 pm
Where: McEnery Convention Center
San Jose, CA.
Saturday Advanced Education Conference Program is COA-Approved for CEs.
Registration includes:
• Business, Wellness and Science Track and Hands-on Workshops. See Program here
• Advanced Education Networking Luncheon (Saturday) with Eva DeVirgilis
• Complimentary 2-day Expo Hall Pass (Sunday & Monday)
• Two days of Supplier Class Sessions (Sunday & Monday)
• Flashdrive of Conference presentations/handouts
• Complimentary Records Verification for all Conference Attendees ($175.00 Savings)
Become Nationally NCEA Certified - Learn more www.NCEACertified.org
Early Bird Rate: Until June 15 - $230 - Register now
June 16 - August 19th - $285 and Onsite Registration is $335.00
Where is your Membership Certificate?
I just noticed at another spa that they displayed their association membership certificate! I thought it was absolutely wonderful! We are professionals, just like doctors, lawyers, etc. so why shouldn't we? It also sends a message to our clients that we show pride in our profession. I'm hanging mine up tomorrow! I challenge all estheticians to get out their Membership Certificates, NCEA Certified credentials and hang them for all their clients to see!
p.s. Most states already require your esthetician license and a facility license be on the wall. Not sure? Check with your state regulatory board http://www.ncea.tv/find-your-state-regulatory-board
Got 10 minutes? Watch this webinar NOW
Your future may depend on it!
Protecting your future as an Esthetician is not just about making money. Knowing and learning what your professional licensing, state regulatory boards and legislators are doing, or NOT doing is key to ensuring the future of our profession!
Post your NCEA Membership Certificate and wear your NCEA Certified pin and...
Bookmark your State Regulatory Board's website in your browser and check it monthly for:
- i) Board meeting dates and try to attend;
ii) Meet your State Regulatory Board members;
iii) Board minutes (if you didn't attend meeting);
iv) Review any press releases (if available);
iv) Bi-annually - Download & Review Rules & Regulations
*Check for revisions & dates - Download & Review Statutes
Learn what other things you should be doing to be involved - Go to Political Advocacy
Missed an issue? Check out the archives and our searchable article database. Click here
- About NCEA
- Member Center
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Unleash your little one's musical side with this Discover & Play Piano from Baby Einstein. Your baby will love touching its soft, responsive keys and discovering its three fun play modes. In instrument mode, choose the drum, violin or horn to hear the keyboard make those sounds. In numbers mode, hear the piano say numbers in English, Spanish or French. And in animals mode, hear the piano mimic dog, cat and duck sounds. This vibrant piano toy includes more than 20 classical melodies and fun sounds, and the instrument buttons light and flash while the melodies play.
Manufacturer's Suggested Age: 3 Months and Up
Number of Pieces: 1
Includes: keyboard
Material: plastic
Product Weight: 1.150
Battery: required, included: 3 AA
other Info.
Warranty Information
To obtain a copy of the manufacturer's warranty for this item, please call Target Guest Services at 1-800-591-3869.
- Online Item #: 14047828
- Store Item Number (DPCI): 030-10-0560
- Item can be gift wrapped.
- Imported


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Ostensibly called by FIA president Jean Todt to discuss regulatory cost control, the meeting scheduled for May 1 at Formula One Management's Biggin Hill headquarters will severely test the executive powers of Formula 1's Strategy Group, and, by extension, the body's future.
Formed as part of the sport's post-2012 governance procedure, the Group consists of the FIA (six votes), FOM (ditto) and four so-called 'Championship Constructors Bonus' teams - Red Bull, Ferrari, McLaren and Mercedes - plus Williams (through heritage) and Lotus, as reward for it being highest-placed non-CCB team in 2012-'13.
As an aside, one wonders which team will be awarded the sixth seat come 2015 should the black/gold operation continue its current downward spiral. As things presently stand, the sixth seat at the table is not allocated permanently, and on present form Force India should receive the call in 2015. However, given the team's relentless campaign against what deputy team principal Robert Fernley calls "disenfranchisement" - see below - the call may not materialise.
Get back on track. Join today for unlimited access to all Autosport news and features.
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South African-born Dieter trained as industrial engineer before holding down a variety of senior motor industry marketing and manufacturing positions. At the age of 40 he decided to follow his passion, and became the first and only South African journalist to cover Formula 1 regularly. Dieter joined AtlasF1 at the beginning of 2004 – a year prior to its merger with Autosport – and his regular column offers an intriguing analysis of F1's politicking and commercial chicanery. Although now also proudly Belgian, he gives his domicile as "Wherever F1 duplicity lurks".@RacingLines More features by Dieter Rencken


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23 Reviews
What an excellent and easy to make stuffing. I used the Roasted Vegetable flavor Ritz's the 2nd time I made this and it was even better. Wish I could give more than 5 stars. Thank You for sharing!
This dish was absolutely delicious! I used unsalted butter, my preference, added some mushrooms and used lump crabmeat. Followed the rest of the recipe. Will be making this dish again. YUM.......
Easy to make and delicious! Made as directed, but added a few minutes of broiling at the end to crisp up the topping and finish cooking of fish....
Perfect! Supper easy can be made healthier by cutting the butter in half without affecting the taste. THe prep is supper easy and the results and dont require much attention.
Aunt Linda, I made this for our special Valentine's dinner, as I have been eying it, and it looked like it would be the perfect entree for my special Valentine's dinner to present to Mike on our special evening together. He took one bite and said okay, this beats my dinner that I made you last night by a mile! Where did you get the recipe? When I told him it was one of your recipes on Recipezaar, he was so happy that I had chosen one of your recipes!! He is always bugging me to come on here and look around, so when I actually did and made one of your recipes he was estatic! You have no idea how much he brags about what a wonderful cook you have always been. I guess I had to see for myself. After having this I have to say, I can learn a thing or two from you in the cooking department! This is the best haddock dish I have ever prepared or eaten, and that's no kidding! My dad would love this and I plan to make it for him and my sisters and have them over soon for dinner. This dish is rich, comforting, moreish and amazing! I can't remember when I have had a better meal in my life. Thank you! Cassie, AND Mike thanks you too :)
I made a half-recipe of this, cutting everything in half except the crab, which I used the whole can. This was very good; definitely a company-worthy dish.
i used Phillips lump crab meat and rolled the fillets around the stuffing. finished under the broiler. tasty for sure!
I needed a quick recipe for a last minute dinner and this was perfect. I had two very thin filets of flounder to work with and no idea how to kick them up. This recipe suited that need perfectly. I took one filet and layed it in the pan (sprayed with a bit of Pam first) and then some stuffing, layed the other filet on and more stuffing. I baked perfectly. Not soggy at all. I broiled it in my toaster oven for just a minute to get the crispy crunch on top. We enjoyed this very very much. Will be doing again. Bet this would be good stuffed inside Snapper!! Yum!
Yummy! I used flounder which was light and delicate. I think next time I might try sprinkling the top of the fish with some Old Bay to "pep" it up a bit. I think I would really like this on salmon. Great find! Thank you, Linda!
This was okay. I used one lemon and then decided mid-dinner that it could use more, so I squeezed a second lemon on top, but it still seemed to be missing a little something for me. Maybe a little white wine and lemon with the fish instead of just the butter. I will try that next time. The stuffing was a little mushy for me also. I stuck it under the broiler for 2 minutes after it came out of the oven and that crisped up the top a little more.


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Everything was great ,went from the 23 - january 3rd . The food was amazing , the people was all beautiful . The drinks was all high class . Oh god speechless cant wait to go back . My family all loved it , we were a group of 20 and we all enjoyed it . The disco was great , the P.O.P was super friendly . This was my 3rd time coming and i felt in love once again . The lobby was all brighten up with lights . The santa claus coming down in a helicpter was a great show . Speechless . The food was definently a huge pluss . Everything so clean and sanitary . had an amazing time . Cant wait to be back . Vamos a volver otra vez al paraiso , - Severino Family .
- Official Description (provided by the hotel):
- The Grand Palladium Palace Resort Spa & Casino enjoys a privileged sea-front location on the renowned Playa Bavaro. The resort, located on a sprawling estate of lush tropical vegetation and a coconut plantation, is next to the Grand Palladium Bavaro Resort & Spa and the Grand Palladium Punta Cana Resort & Spa. The facilities of these adjoining resorts are open to guests of the Grand Palladium Palace Resort Spa & Casino. ... more less
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- Also Known As:
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- Palladium Punta Cana


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FASEB MARC Student Development & Mentoring Programs
Career Services and Professional Development Programs
"FASEB Row" at scientific meetings
FASEB MARC Peer Mentors Network
FASEB MARC Travel Awards available for scientific meetings
FASEB MARC Passport to Success
FASEB MARC Student Day activities at scientific meetings
FASEB MARC Career Development Services at meetings
Grantsmanship Training for PhD Students/Postdocs & Junior Faculty
EB2016/FASEB Career Center Companion App is now mobile! Career Center agenda, post jobs, Facebook, Twitter, alerts, more on your iPhone/Android, free! Get our new Guidebook http://guidebook.com/g/EB2016Careers/
The Poster/Platform Presenter Travel Award Application forms for scientific meetings held in 2016 are now available. Visit


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Felicia Farr (born October 4, 1932) is a former American actress and model....
Connect any celebrity with Felicia Farr to see how closely they are linked... romantically!
I just thought I`d remark on a movie I watched today called The Last Wagon and I saw real beauty in your performance as well as in your appearence
Felicia- I knew you when we were children- your mother was Sylvis- sn electrolgidt- we lived in Maspeth- my mother was Nan Berger- does this sound familiar at all?? we always had to watch for you sitting in the audience of the Academy awars- cheers


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Pitbull with Ke$haTue, June 4, 2013 - 8:00 PMBell Centre in Montreal, QC
We are sorry, the date for this event has already passed. This event took place on 6/4/2013. Click here to view upcoming events for Pitbull with Ke$ha.
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hr representative jobs near San Antonio, TX
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Human Resources Generalist
Visionworks - San Antonio, TX
Act as back up to HR Specialist for union job postings. The HR Generalist will be responsible for administering various human resources support programs to...
Commercial Lines Customer Service Representative
Nationwide Mutual Insurance Company - San Antonio, TX
HR and Director. Learns to service commercial insurance business via direct (inbound) telephone and internet contact with current customers in multiple states....
Human Resources Associate
Visionworks - San Antonio, TX
One to three (3) years of HR experience or related field experience. First contact for all HR administrative issues for stores and departments in service area;...
Outside B2B Sales Representative
Paycom - San Antonio, TX
This position is a great fit for someone who is interested in meeting with top-level Executives every day and helping them to optimize their current HR and...
Bilingual Customer Service Representative
Conduit Global - San Antonio, TX
$12.50 hr after training. Conduit Global is now Hiring Bilingual Customer Service Representatives. Customer Service Representatives performance will be measured...
Human Resources Director - Principle Auto
Principle Auto - San Antonio, TX
Prepares government reports related to EEO compliance or other HR functions. Acts as liaison with various insurance carriers/brokers and fosters effective...
Sales Apprentice
ADP - San Antonio, TX
Support Outside Sales Representative with developing and executing a cold calling strategy to target prospects for ADP payroll and HR solutions....
Staffing Manager
Elwood Staffing - San Antonio, TX
Serve as customer service representative to client companies and. Manager, Customer Service Manager, HR Manager, Human Resources....
Manager, Technical Call Center - RG
Absolute Opportunities - San Antonio, TX
Responsible for establishing and communicating process and goals, reviewing reported results and KPIs to improve productivity, highlight deficiencies and drive...
Earn $11-$16 / hr. We'll train you to become a Pharmacy Tech!
Medix - San Antonio, TX
Retail sales, cashiers, bartenders, customer service representatives, CSR, servers, administrative assistants Requirements....


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(This story first ran at PressConnects.com) BALTIMORE — At 26 years old, UFC light-heavyweight champion Jon Jones admits it is difficult even for him to realize he could soon be considered the best fighter in the history of mixed martial arts. But it's a reality (…)
Coming off two impressive stoppages, Ariel Sexton is looking to make a name for himself by taking out UFC veteran Roger Huerta at ONE: Dynasty of Champions. read news >>