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Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.
Author Summary
Natural killer (NK) cells are part of the innate immune response against virus infection and cancer. Recently we demonstrated that ligands for the NK cell activation receptor, NKG2D, trigger NK cell-mediated response to infected cells. These ligands are expressed on HIV-1-infected cells and not on uninfected cells. Despite the observation that NKG2D ligands are expressed on infected cells, it is unclear how HIV-1 induces their expression. In the present study, we demonstrate that HIV induces the ligands of the NKG2D receptor through the viral gene product Vpr. Vpr triggers a DNA damage response in infected cells, which in turn, increases virus production. We also demonstrate that by blocking the activity of ATR, a major component in the DNA damage response, we were able to prevent NKG2D ligand expression. When Vpr was removed from the virus genome, NK cells lost their ability to lyse the HIV-infected cells. Thus, HIV-1 actively triggers NK cells through the activity of its viral gene product, Vpr.
Citation: Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, et al. (2009) HIV-1 Vpr Triggers Natural Killer Cell–Mediated Lysis of Infected Cells through Activation of the ATR-Mediated DNA Damage Response. PLoS Pathog 5(10): e1000613. https://doi.org/10.1371/journal.ppat.1000613
Editor: Thomas J. Hope, Northwestern University, United States of America
Received: October 7, 2008; Accepted: September 11, 2009; Published: October 2, 2009
Copyright: © 2009 Ward 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 supported by grants from NIH/NIAID (http://www3.niaid.nih.gov/): AI52809 and AI56923 for EB and AI49057 for VP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
NK cells are involved in the immune response against tumor cells and virus-infected cells without the requirement for previous exposure to their targets or their products. The importance of NK cells in restraining viral infection was first shown in studies using murine models in which NK cells were depleted [1],[2],[3]. In these studies, NK cell depletion led to enhanced viral replication and cytopathology. In humans, NK cells control the severity of viral infections such as those by herpes simplex virus [4],[5], cytomegalovirus [6] and hepatitis B virus [7]. Lack of NK cells in humans or defects in NK cell function are associated with fatal disseminated herpes virus infection [8],[9],[10],[11],[12],[13].
One of the major roles of NK cells in controlling viruses is the destruction of the infected cells. Direct target cell killing by NK cells is mediated by the regulated release of granules containing perforin and granzymes [14],[15]. Perforin forms pores in the plasma membrane allowing ions and small particles into and out of the cell [16]. The granzymes most likely enter the target cell through perforin-formed channels [17] or endocytosis [16] and induce apoptosis of the infected cells. NK cells may also kill through engagement of death receptors (e.g., Fas) by ligands expressed on the NK cell surface (e.g., CD178) whose surface expression is increased upon degranulation [18]. In vivo, cytotoxic function is primarily mediated by the subset of NK cells that are CD56dim/CD16pos, the dominant subset in the peripheral blood of healthy individuals [19]. CD56bright/CD16neg cells constitute a minor NK population in the blood but are the major NK cell subset in secondary lymphoid tissues. CD56bright NK cells express less perforin than CD56dim NK cells but higher concentrations of cytokines and as such are important in regulating immune responses.
NK cell release of cytotoxic contents is regulated by a large array of signals provided by a variety of membrane-bound, activating receptors expressed on NK cells that interact with their corresponding ligands on target cells (see [20] for a current list of all known activating receptors and their ligands). Almost all NK cell activating receptors interact with adaptor proteins containing activation domains [20]. Activation receptors can be divided into those associated with intracellular tyrosine activation motifs (ITAM)-containing adaptor molecules and those associating with adaptor molecules lacking ITAMs. ITAM-dependent receptors include: CD16 (low affinity IgG receptor), and the natural cytotoxicity receptors (NCRs; NKp30, NKp44 and NKp46) [21]. The NKG2D receptor is an example of an ITAM-independent activation receptor. NKG2D is found on almost all peripheral blood NK cells and is a member of the NKG2 receptor family. NKG2D does not associate with CD94, but forms homo-dimers on the membrane of NK cells [22],[23]. Although NKG2D specifically recognizes its ligands on target cells and is important for activation of NK cells, it does not induce signal transduction within the cells. For this purpose, NKG2D associates, on the membrane, with the DAP10 adaptor molecule. The DAP10 adaptor protein contains a YXXM motif instead of ITAM and recruits phosphatidylinositol-3- kinase and Grb-2 upon phosphorylation [20].
Our prior studies [24],[25] and those of others [26] indicate that HIV-1-infection of primary CD4pos T-cells leads to the surface expression of NKG2D ligands. The ligands for human NKG2D are the MIC-A and -B and ULBP 1–4 [27],[28]. ULBPs but not MIC-A or -B were found on the surface of HIV-infected cells in our prior studies. We not only found these molecules on in vitro infected primary CD4pos T-cell blasts but also on infected cells obtained from HIV-1-infected patients after amplification of the virus-infected cells ex vivo [25]. In addition, these studies, which used primary CD4pos T-cells infected with HIV-1 as targets for autologous NK cells in cytotoxicity assays, revealed that NK cells can respond to the HIV-1-infected cells in an NKG2D-dependent manner [24],[25].
A recent study by Gasser et al. demonstrated, that DNA damage induced expression of a plethora of NKG2D ligands [29]. The up-regulation of NKG2D ligands occurred after the activation of the DNA damage recognition enzymes ataxia telangiectasia mutated (ATM) and ATR [29]. The HIV-1 Vpr protein is a potent activator of ATR, and induces infected cells to arrest in the G2 phase of the cell cycle, [30],[31] and increases viral transcription from the HIV-1 long-terminal repeat [32],[33],[34]. Our previous studies have demonstrated that the ability of Vpr to induce cell cycle arrest in G2 is dependent on activation of ATR but not ATM [30],[35]. Therefore, we hypothesized that HIV-1 Vpr is responsible for inducing the expression of NKG2D ligands during infection through activation of ATR. We further hypothesized that Vpr-mediated up-regulation of NKG2D ligands would lead to NK cell activation. If these hypotheses are correct, this would indicate that the DNA damage signaling by Vpr has consequences that may potentially be detrimental to the virus because they enhance immune surveillance by NK cells.
Vpr induces expression of the NKG2D ligands, ULBP-1 and ULBP-2
Initially, we determined the role of Vpr in the up-regulation of NKG2D ligands on primary CD4pos T-cells. To eliminate possible differences in replication kinetics due to the presence or absence of Vpr [32],[33], we used a defective HIV-1 construct, DHIV, that has a deletion in the env gene. We then provided the VSV-G glycoprotein in trans, to form pseudotyped virions [36],[37]. In humans, six different NKG2D ligands have been reported to date. In order to be able to detect global changes in the expression of all six NKG2D ligands, we utilized a recombinant soluble NKG2D receptor that binds to all of them. We stained the cells with viability dyes and evaluated only the viable infected population for expression of NKG2D ligands (Figure S1).
We tested the binding of soluble NKG2D as a measure of ligand expression in PBMC from five individuals, in the presence or absence of in vitro DHIV infection. As seen in Figure 1A, CD4pos T-cells infected with DHIV wild type (WT) expressed NKG2D ligands [MFI = 687 for infected cells (HIV-1 p24 Agpos/CD4neg) compared with MFI = 159 for uninfected control]. Experiments were performed in parallel for a total of 5 donors and the statistical difference in MFI between the infected and uninfected groups in five individuals was p<0.01 based on the Student's t-test. Uninfected cells did not detectably express NKG2D ligands (MFI = 152 for uninfected cells compared with MFI = 159 for secondary Ab staining). Within the infected population, only the HIV-1 p24 Agpos cells, but not the p24neg in the same culture, expressed NKG2D ligands (see Figures 1 and S2). Therefore, we conclude that NKG2D is not induced on uninfected, bystander cells. DHIV does not encode the envelope glycoprotein; however NKG2D ligands are also induced on envelope-expressing HIV-1NL4/3-infected cells (Figure 1B). DHIV is derived from HIV-1NL4/3. Thus, DHIV-infected cells express NKG2D ligands on their cell surface.
DHIV (A) and HIV-1NL4/3 (B) -infected primary T-cell blasts and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 antigen (Ag). Histograms were derived following acquisition on a flow cytometer of either 104 viable cells [(for uninfected cells) blue line] or 104 viable CD4neg cells and HIV-1 p24 Agpos infected cells (red line). As controls (green line), cells were stained with secondary Ab alone. This figure is representative of five separate experiments.
We then sought to determine whether Vpr was responsible for the expression of NKG2D ligands. We generated DHIV containing a truncation in Vpr. As controls, mutants of DHIV unable to express Vif, Vpu or Nef were also generated. Figures 2 and S2 illustrate that the DHIV-ΔVpr infected cells failed to induce NKG2D ligand expression (Figure 2B; MFI = 188 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 216 for HIV-1 p24 Agneg/CD4pos infected cells). The MFI for DHIV-WT infected cells was statistically different (p<0.01) compared to the MFI for DHIV-ΔVpr infected cells. We compared expression of NKG2D ligands on CD4pos T-cells infected with DHIV-ΔVif (Figure 2C; MFI = 600 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 260 for HIV-1 p24 Agneg/CD4pos infected cells), DHIV-ΔVpu (Figure 2D; MFI = 777 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 247 for HIV-1 p24 Agneg/CD4pos infected cells), and DHIV-ΔNef (Figure 2E; MFI = 698 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 303 for HIV-1 p24 Agneg/CD4pos infected cells). Therefore, expression of NKG2D ligands induced by ΔVif, ΔVpu and ΔNef DHIV was comparable that induced by WT DHIV (Figure 2A). Thus, we conclude that Vpr is required for HIV-1-mediated up-regulation of NKG2D ligands on the surface of infected cells.
Infected primary T-cell blasts [(A) Wild-type, (B) ΔVpr, (C) ΔVif, (D) ΔVpu and (E) ΔNef] and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 Ag. Histograms were derived following acquisition on a flow cytometer of either 104 viable CD4pos cells [(for uninfected cells) blue line] or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). The figure is representative data from three separate experiments.
To determine whether Vpr expression is sufficient to induce NKG2D ligands, we resorted to a lentiviral vector that encodes HIV-1 Vpr and GFP but no other viral gene (pPR-VIP). As a control, we used a similar lentiviral vector encoding only GFP. We observed that Vpr alone, but not the control lentiviral vector, was able to induce NKG2D ligands on CD4pos T-cells (Figure 3). Therefore, Vpr is sufficient for HIV-1 to induce NKG2D ligands on the infected cell surface.
Primary CD4pos T-cell blasts were transduced with a lentivirus vector (pPR-Vip) coexpressing Vpr and GFP (red line). As a control the same cells were infected with a lentivirus vector expressing GFP only (green line). Following transduction cells were stained with a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. Histograms were derived following acquisition on a flow cytometer of either 104 viable cells [(for untransduced cells) blue line] or 104 viable GFPpos cells (green and red line). The figure is representative data from two separate experiments.
The studies illustrated in Figures 1–3 utilized a soluble NKG2D construct that is unable to distinguish between the various ligands. Hence, we wished to determine which of the NKG2D ligands were specifically induced by Vpr. For this purpose, we compared the expression of ULBP-1, ULBP-2, ULBP-3, MIC-A and MIC-B on DHIV-infected cells. In the studies shown in Figure 4, we demonstrate that WT virus induced ULBP-1 (Figure 4A; MFI = 483 for WT virus-infected cells compared with MFI = 167 for uninfected cells) and ULBP-2 (Figure 4C; MFI = 539 for WT virus-infected cells compared with MFI = 131 for uninfected cells). In contrast, little or no induction of ULBP-3 (Figure 4E; MFI = 150 for WT virus-infected cells compared with MFI = 134 for uninfected cells), MIC-A (Figure 4G; MFI = 187 for WT virus-infected cells compared with MFI = 106 for uninfected cells) or MIC-B (Figure 4I; MFI = 99.8 for WT virus-infected cells compared with MFI = 90.5 for uninfected cells) was detected.
Primary CD4pos T-cell blasts were infected with wild-type (WT) HIV-1. As a control, the same cells were infected with HIV-1 that was deficient in expression of Vpr (ΔVpr). As an additional control we evaluated uninfected CD4pos T-cells for expression of individual NKG2D ligands (blue lines). Following infection, cells were surface stained using fluorochrome-conjugated mAb specific for: CD4 (A-J), ULBP-1 (A–B), ULBP-2 (C–D), ULBP-3 (E–F), MIC-A (G–H) or MIC-B (I–J). The cells were then intracellularly stained for HIV-1 p24 antigen. Histograms were derived following acquisition of 104 viable uninfected CD4pos cells (blue line) or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). Green line represents the histogram of staining controls (isotype controls). The figure is representative data from two separate experiments.
DHIV-ΔVpr induced considerably lower levels of ULBP-1 (Figure 4B; MFI = 220 for ΔVpr virus-infected cells compared with MFI = 485 for WT virus-infected cells) and ULBP-2 (Figure 4D; MFI = 179 for ΔVpr virus-infected cells compared with MFI = 539 for WT virus-infected cells) than WT virus. Thus, HIV-1 primarily induces the expression of ULBP-1 and ULBP-2 through Vpr.
Induction of ULBP-1 and ULBP-2 by Vpr occurs at the level of gene expression
Next we asked whether induction of NKG2D ligands on infected cells was accomplished through increased steady-state levels of ULBP-1 and ULBP-2 mRNA. We infected CD4pos T-cells with DHIV and select mutants, and then measured the mRNA levels of ULBP-1, ULBP-2, ULBP-3, MIC-A and MIC-B relative to the level of GADPH mRNA (Figure 5). We found that both ULBP-1 and ULBP-2 mRNA levels were 15-fold higher in WT virus-infected cells compared with ULBP-1 and ULBP-2 gene products in uninfected cells. In comparison to WT DHIV-infected cells, DHIV-ΔVif-infected cells showed little or no changes in ULBP-1 and ULBP-2 mRNA levels. In addition, in DHIV-ΔVpr-infected cells, the levels of ULBP-1 and -2 mRNA were 3 to 10-fold lower compared with those in WT DHIV-infected cells (Figure 5). Infection with WT DHIV only induced a 1.5-fold increase in the expression ULBP-3, MIC-A and MIC-B. Thus, among the five NKG2D ligands we evaluated, Vpr is responsible for up-regulating the levels of ULBP-1 and ULBP-2 mRNA in HIV-1-infected cells.
RNA was extracted from HIV-infected CD4pos T-cell blasts and uninfected cells and reverse transcribed. Relative copy numbers of NKG2D ligands were determined by real-time PCR using NKG2D-ligand specific primer pairs and normalized relative to GAPDH expression. The relative increase in copy numbers was calculated as described in Materials and Methods. The relative copy numbers for the indicated primer pair in uninfected cells was set to 1. Error bars indicate the standard deviations of the mean of samples in triplicate.
Expression of NKG2D ligands requires Vpr's ability to interact with the Cullin 4aDCAF-1 E3 ubiquitin ligase and to activate ATR
Vpr induces G2 arrest of infected cells through interaction with a Cul4a-based ubiquitin ligase that also contains the adaptor, DDB1, and the substrate receptor, DCAF1 (reviewed in [38]). Despite the fact that the degradation target for this ubiquitin ligase is unknown, the consequences of the Vpr-E3 complex are well documented. The main result is the activation of the ATR kinase [39] that, in turn, leads to G2 arrest [35]. Therefore, we wished to determine whether Vpr recruitment of the Cullin-4a-based E3 ubiquitin ligase complex is required for ULBP-1 and -2 expression.
We first tested whether domains in Vpr that are involved in recruiting or activating the Cullin-4a-based E3 ubiquitin ligase were required for induction of ULBP-1 and ULBP-2. The Vpr Q65R and Vpr R80A mutations have been previously shown to abate the ability of Vpr to induce cell cycle arrest [39],[40]. Vpr Q65R is unable to bind to DCAF1; the exact defect induced by R80A mutation is unknown, and it has been proposed that R80A abates the interaction between Vpr and the ubiquitination target for the E3/Vpr complex [39],[40]. For this purpose, we generated DHIV mutants with the above substitutions in Vpr. As shown in Figure 6, expression of NKG2D ligands was diminished by either substitution, although Vpr(R80A) had a more dramatic effect than Vpr(Q65R) (18 percent of wild-type Vpr and 48 percent of wild-type Vpr, respectively). The cell cycle profiles were examined in infected cells to visualize functional deficits in these mutants. When Vpr R80A or Vpr Q65R, were expressed neither mutant virus could induce G2 arrest as effectively as WT virus (Figure S3).
Primary T-cell blasts infected with wild type HIV-1 and HIV-1 with various mutations in Vpr were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. Uninfected CD4pos T-cells were surface stained in a similar fashion. All cells were stained intracellularly for HIV-1 p24 Ag. The mean fluorescent intensity of NKG2D ligand staining was obtained from collection of 104 CD4neg p24pos cells for all infected cells, and of the 104 CD4pos p24neg cells for the uninfected control. This figure is a representative of three separate experiments.
To more directly assess the requirement of the Cullin-4a-based E3 ubiquitin ligase in inducing NKG2D ligand expression, we resorted to RNA interference-mediated depletion of DCAF1 (Figure 7). Results shown in Figure S4 demonstrate that infection with HIV-1 and indicated mutant viruses had no effect on the levels of DCAF1 protein. We generated a lentivirus vector, based on FG12 [41], expressing short hairpin RNAs (shRNAs) specific for DCAF1 [42]. Knockdown of DCAF1 expression completely abated the ability of WT DHIV to up-regulate NKG2D ligand expression (Figure 7C) compared with the same infected cells expressing a control (scrambled sequence) shRNA (Figure 7B) or untransduced cells (Figure 7A). Thus, NKG2D ligand expression on HIV-1-infected cells is dependent on the Cul4aDCAF-1 ubiquitin ligase complex.
Primary CD4pos T-cell blasts were transduced with shRNA of a DCAF1-specific sequence (C) or an shRNA with a scrambled sequence (B). Untransduced cells were used as a negative control (A). The cells were then infected and stained with anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. Transduced cells were detected by GFP expression. The figure is representative data from two separate experiments.
Manipulation of the Cul4aDCAF-1 complex by Vpr results in ATR activation and G2 arrest [37]. It is formally possible that up-regulation of ULBP-1 and -2 is also a consequence of manipulation of Cul4aDCAF-1, but independent of ATR activation. Alternatively, ATR activation may be required for ULBP up-regulation, as was suggested by Gasser et al [29]. To differentiate between these two possibilities, we treated DHIV-infected cells with caffeine, a known inhibitor of ATR and its related kinase, ATM [30],[35],[43]. As shown in Figure S5, caffeine, at a concentration of 4 mM, eliminated the ability of HIV-1 to induce G2 arrest from a ratio of G2+M/G1 = 7.67 to ratio of G2+M/G1 = 0.53. Caffeine (4 mM) treatment of HIV-1-infected cells dampened NKG2D ligand expression (Figure 8; MFI = 277 for caffeine-treated virus-infected cells compared to MFI = 763 for vehicle-treated virus-infected cells). Caffeine had little effect on NKG2D ligand expression on uninfected cells (Figure 8; MFI = 148 for caffeine-treated uninfected cells compared to MFI = 103 for vehicle-treated uninfected cells). Thus, caffeine inhibition of ULBP up-regulation suggests that ATR is a required upstream mediator of NKG2D ligand expression.
Primary CD4pos T-cells were infected in the presence (C) or absence (B) of the ATR inhibitor, caffeine. As controls CD4pos T-cells were not infected (A) but treated with caffeine or vehicle. Following exposure to 4 mM caffeine and HIV-1 infection, NKG2D ligand expression on the surface of infected cells was measured by staining with a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. The histograms are gated for either 104 viable CD4pos/HIV-1 p24neg cells (blue) or on 104 viable CD4neg HIV-1 p24pos cells (red). This figure is representative of three separate experiments.
Since caffeine inhibits both ATR and ATM [43] it is formally possible that the effect of HIV Vpr on ULBP up-regulation is dependent on both ATR and ATM. Thus, to determine any role that ATM may play in the induction of NKG2D ligands by HIV, we evaluated the effect of KU55933, an ATM-specific inhibitor on NKG2D ligand expression [44]. Figure 9 shows that treatment of infected cells with 10 µM KU55933 had little effect on NKG2D ligand expression (MFI = 953) relative to infected cells treated with vehicle alone (MFI = 955). In contrast, KU55933 did affect the level of NKG2D ligands expressed on primary CD4pos T-cells following treatment with aphidicolin, a DNA polymerase α inhibitor which induces replication stress (MFI = 432) relative to vehicle-treated cells (MFI = 792) (Figure S6). Unlike KU55933, caffeine prevented NKG2D ligand expression on both HIV-1-infected and aphidicolin treated cells (see Figures 9 and S6). Thus, Vpr induces NKG2D ligand through activation of ATR but not ATM.
Primary CD4pos T-cells were infected in the presence (B) of the ATM-specific inhibitor KU55933 (10 µM). As a positive control infected cells were treated with 4 mM caffeine (C). Negative controls included vehicle-treated infected cells (A) or CD4pos T-cells infected with HIV-1 lacking Vpr [(ΔVpr) D]. Following exposure to inhibitors and HIV-1 infection, NKG2D ligand expression on the surface of infected cells was measured by staining with a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. The histograms are gated for either 104 viable CD4pos/HIV-1 p24neg cells (blue) or on 104 viable CD4neg HIV-1 p24pos cells (red). MFI = mean fluorescent intensity. This figure is representative of three separate experiments.
Up-regulation of ULBP 1 and 2 by Vpr contributes to killing by NK cells
Our studies, thus far, demonstrate that HIV-1 induces the expression of ULBP-1 and ULBP-2 through Vpr. However, whether Vpr mediated-induction of ULBP-1 and ULBP-2 on infected cells constitutes a signal that will trigger NK cell lysis remains to be determined. To address this, we compared the ability of primary NK cells to lyse autologous T-cell blasts when infected with either WT or ΔVpr viruses. As a control, we blocked NKG2D on NK cells prior to exposure to target cells. If NKG2D interaction with its ligands on infected cells had any role in triggering NK cell lysis, then blocking NKG2D would abate the effect. As shown in Figure 10, cells infected with WT virus are sensitive to NK cell lysis, when compared with uninfected cells. Infection with DHIV-ΔVpr led to a reduced level of NK-mediated lysis (Figures 10 and S7). Although the reduction was not complete, it is noteworthy that deletion of Vpr had a similar effect as the blockade of NKG2D (Figure 10A). These observations, taken together, indicate that up-regulation of NKG2D ligands by Vpr is responsible for a fraction of the observed lytic activity by NK cells. Thus, we conclude that through its known abilities to interact with the Cullin 4a-based E3 ubiquitin ligase and activate ATR, Vpr induces expression of ULBP-1 and -2 and this, in turn, constitutes a signal that triggers NK cell lysis of infected cells.
Primary CD4pos T-cell blasts were infected with HIV-1 that was deficient in expression of Vpr (ΔVpr) (A&B). As a control, the same cells were infected with wild-type (WT) HIV-1 (A&B). Following infection the infected cells were isolated, labeled with 51Cr and mixed with autologous NK cells at 2.5∶1 (open bars) and 5∶1 (closed) effector cell to target cell ratios. Each group was done in triplicate. Prior to the lytic assay some of the NK cells were exposed to blocking antibodies to NKG2D (A). At the end of the incubation period culture fluids were harvested and analyzed for the presence of 51Cr. Percent specific lysis was determined as described in the Materials and Methods section. Error bars represent standard deviation of the mean. The figure is representative of two separate experiments. *p<0.05, **p<0.01 by the Student's t-test.
Our previous studies demonstrated that primary T-cell blasts infected both in vitro and in vivo with HIV expressed, on their cell surface, ligands for NKG2D [24],[25]. Here, we demonstrate that HIV-1 Vpr selectively induces expression of ULBP-1 and ULBP-2 gene products (Figures 4 and 5). ULBP-3, MIC-A and MIC-B were hardly, if at all, up-regulated. We did not observe ULBP-4 on HIV-infected cells using a recently generated anti-ULBP-4 monoclonal antibody (a gift from Dr. John Trowsdale, University of Cambridge, Cambridge, England) in a separate experiment (data not shown).
DNA damage leads to the expression of NKG2D ligands [29]; our observations here indicate that Vpr, through activation of the DNA damage pathway, induces similar effects as those reported by Gasser [29]. This ultimately leads to expression of ULBP-1 and -2 on the cell surface. Despite the potential abilities of both ATM and ATR, following DNA damage, to induce NKG2D ligands [29], only ATR in HIV-1 infected cells appears to trigger the expression of NKG2D ligands. This is consistent with the previously reported specificity of Vpr for ATR and not for ATM [30],[31]. We now show that Vpr enhances NKG2D ligand expression on infected cells and that ATR is required for this novel activity of Vpr (see Figures 8 and 9).
The available literature on whether retroviruses activate DNA damage responses upon integration remains controversial. Several publications propose that HIV (and other retroviruses) do not cause DNA damage signaling when they integrate [45],[46] while others have shown the participation and/or requirement of checkpoint proteins and DNA damage sensors [47],[48]. HIV-1 Vpr activates the DNA damage sensor, ATR, but importantly, Vpr does not do so upon integration, but only after de novo expression in the infected cells, well beyond the integration step [46]. Furthermore, we found no evidence that the presence of Vpr in virions early in infection enhanced the efficiency of viral integration to any degree [46]. Accordingly, Vpr has been shown to exert identical effects on the cell cycle when expressed via systems devoid of viral integration (such as adenoviral vectors, plasmid transfection, and tetracycline-inducible Vpr cell lines [49],[50],[51]).
Recent studies from other investigators indicate that soluble Vpr has an impact on NK cell activity [52]. Although we did not directly assess the role of soluble Vpr, our observations produced no evidence that soluble Vpr released by HIV-infected cells would induce NKG2D ligands on uninfected cells. We base this notion on the fact that uninfected (HIV-1 p24neg) cells within infected cultures do not express NKG2D ligands on their surface. Only HIV-1 p24pos cells express NKG2D ligands (see Figures 1 and S2). Hence, we propose that Vpr must be expressed within the infected cells in order to induce NKG2D ligand expression.
HIV-1 accessory gene products other than Vpr appear to have no effect on the expression of NKG2D ligands. Our observations here are in contrast to those of Cerboni et al. who showed that HIV-1 Nef down-regulates ULBPs from the cell surface of Jurkat T-cells [26]. The contradictory observations made in our studies may suggest that there are differences in the ability of Nef to modulate NKG2D ligands between activated primary T-cells and transformed T-cell lines. The ΔNef virus we used in our study does not express Nef even though it expresses Vpr (Figure S8B).
The Cul4aDCAF-1 ubiquitin ligase contributes to the ability of Vpr to arrest HIV-1-infected cells in G2 phase of the cell cycle [38],[39]; in our current studies we showed that this ligase is important in Vpr-induced NKG2D ligand expression as well. E3 ubiquitin ligases transfer ubiquitin molecules to specific substrates. Poly-ubiquitination can mark substrates for degradation by the proteasome. We attempted to test the role of proteasomal degradation in induction of NKG2D ligands. Unfortunately, proteasome inhibitors were highly toxic to primary T-cells and, therefore, the possible role of proteasomal activity in degrading NKG2D ligands is unknown at this time (data not shown).
Our expectation was that induction of cell-surface expression of ULBP-1 and ULBP-2, would trigger NK cells to kill infected cells. This indeed is what we found, since NK cells have a decreased ability to lyse virus-infected cells lacking Vpr. Moreover, the reduction in killing activity of the ΔVpr virus was strikingly similar to that obtained against WT virus-infected cells in which we masked the NKG2D receptor. We have reported, in previous published studies, that variations exist between HIV-infected donors in the capacity of their NK cells to kill autologous HIV-infected cells [25]. We observed killing from as little as 4% and as high as 20% in 8 donors using an E∶T ratio of 10∶1 [25]. Importantly, The level to which NK cells lysed HIV-infected targets directly correlated with the extent to which ULBP2 was expressed on the surface of the infected cells [25]. Those observations provide indirect support to our model that Vpr, through enhancing ULBP expression, may prime cells for lysis by NK.
NK cell function in HIV-infected individuals is similar to that of uninfected individuals unless the patient is highly viremic [53]. These viremic patients typically possess a dysfunctional NK cell subpopulation lacking CD56 but retaining CD16 [54]. Despite the presence of dysfunctional NK cells in viremic individuals, there are subpopulations of functional NK cells in these patients [53] with residual ability to lyse autologous HIV-infected cells [25]. It has been shown that this residual activity required recognition of NKG2D ligands on the infected cell surface [25].
Based on our studies, the expected consequence of Vpr's induction of ULBP-1 and -2 would be that HIV-1-infected cells would become sensitive to killing by NK cells. This scenario would clearly be detrimental to virus replication. A logical explanation for this paradox is that up-regulation of ULBPs by Vpr is a downstream consequence of its biological activity (e.g., Vpr induces LTR transactivation in the G2 phase of the cell cycle [32]). Therefore, we hypothesize that other unknown cellular factors may compensate for the activity of Vpr to collectively modulate the sensitivity of HIV-infected cells to lysis by NK cells.
It is difficult to reconcile how HIV-1 could persist if it increases the likelihood of being destroyed by NK cells through induction of ULBP-1 and ULBP-2 (Figure 4) by Vpr on the cell surface and down modulating ligands (i.e., HLA-A and –B) to NK cell inhibitory receptors by Nef [55]. Part of the answer may lie in the fact that Nef does not down modulate HLA-C and HLA–E [55],[56]. We have published previously [56] that NK cells lacking HLA-C and –E inhibitory receptors kill infected cells by 5-20-fold over unsorted NK cells. Blocking HLA-C and –E on HIV-infected cells from interacting with inhibitory receptors on NK cells enhances NK cells' ability to kill HIV-infected cells [56]. Overall these findings indicate that HLA-C and –E on the infected cells prevent NK cells from killing the infected cells because inhibitory receptors are engaged on NK cells. However, our previous study also indicated that a sufficient number of NK cells lack HLA-C and HLA–E inhibitory receptors and thus would not be inhibited by these MHC class I molecules [55],[56].
The presence of ULBP-1, ULBP-2 on the infected cell surface would allow the subpopulation of NK cells not regulated by HLA-C and HLA–E to kill the HIV-1-infected cells. However, NK cells are also regulated at the level of coactivating receptors [57]. Degranulation by NK cells following the triggering of NKG2D requires simultaneous engagement of co-activating receptors such as 2B4 or NTB-A by their ligands on target cells [57]. Therefore, the relatively low lytic activity induced by Vpr through ULBP-1 and -2 induction could be explained on the basis of lack of concomitant induction of coactivating ligands. Recently we demonstrated that co-activating ligands are important for NK cell destruction of HIV-1-infected cells [24] and that HIV-1 down modulates the coactivating receptor ligands [24],[25]. This would then make the infected cell less vulnerable to destruction since activated NK cells will have a reduced ability to degranulate. We are currently investigating how HIV-1 down modulates the ligands for the NK cell coactivating receptors in order to test this hypothesis.
Our observation that HIV-1 Vpr induces NK cell ligands opens a new set of questions that will need to be addressed in the near future. Specifically, it will be compelling to ascertain whether up-regulation of ULBP-1 and 2 by Vpr can be exploited therapeutically, such that HIV-1 infected cells can be manipulated to become more susceptible to NK lysis; (ii) whether other accessory or, in general, viral proteins can manipulate other aspects of the NK response; and (iii) the signaling steps linking activation of cell cycle checkpoint proteins and the increase in steady-state mRNA levels for NKG2D ligands.
Materials and Methods
Antibodies and fusion proteins
The mouse anti-human CD4, CD16, CD56, CD112 and MIC-A/MIC-B monoclonal antibodies (mAbs) were obtained from BD Biosciences (http://www. bdbiosciences.com/). The soluble fusion protein of the NKG2D receptor with the Fc portion of human IgG and mouse anti-human NKG2D, ULBP-1, ULBP-2, and -3 antibodies (Abs) were obtained from R&D Systems (www.rndsystems.com). The anti-MIC-A and MIC-B mAbs that do not cross-react with one another were obtained from Axxora (www.axxora.com). The fluorochrome-conjugated goat anti-human IgG1 Fc-specific secondary Ab (with minimal cross-species reaction) was obtained from Jackson ImmunoResearch Laboratories (www.jacksonimmuno.com) and the fluorochrome-conjugated rabbit anti-mouse IgG secondary Ab was from Dako (www.dako.com). The mouse anti-HIV-1 p24 mAb, KC57, was obtained from Beckman Coulter (Fullerton, CA) and the mouse monoclonal Ab clone AG3.0 was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (www.aidsreagent.org) and was deposited by Dr J. Allan [58].
Cells and culture reagents
All primary cells used in this study were isolated from peripheral blood drawn from all healthy donors after informed written consent was obtained in accordance with the Declaration of Helsinki and the policies of the Institutional Review Board at Rush University Medical Center, Chicago, IL. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation (1000× g, 20 min 20°C of peripheral blood over a Ficoll-hypaque gradient (Mediatech, http://www.cellgro.com/). CD4pos T-cells were isolated from PBMC by negative selection using a CD4pos T-cell isolation kit (Dynal, http://www.invitrogen.com/) and stimulated with anti-CD3/anti-CD28 mAb-coated microbeads (Dynal) in RPMI complete medium that consisted of RPMI medium (Mediatech) supplemented with 10% heat inactivated (56°C, 30 min) fetal bovine serum (FBS) (Mediatech) and penicillin/streptomycin (Mediatech) before infection with DHIV or transduction with lentivirus vectors. The Jurkat E6-1 cell line was obtained from the American Type Culture Collection (http://www.atcc.org/) and was maintained in RPMI complete medium. The 293 FT cell line (Invitrogen), used in the generation of virus and vectors, was maintained according to the manufacturer's specifications. Caffeine was obtained from the Sigma Chemical Company (http://www.sigma.com/). KU55933 was obtained from Calbiochem (http://www.calbiochem.com). Aphidicolin was obtained from the Sigma Chemical Company. KU55933 and aphidicolin stock solutions were prepared by dissolving the drugs in DMSO (Sigma Chemical Company) at 1 mM concentration prior to their addition to medium at a final concentration of 10 µM.
Virus vectors
The envelope-defective DHIV vector is isogenic to the HIV molecular clone HIV-1NL4/3. To construct DHIV vectors with premature stop-codons in the nef, vpu, vif and vpr open reading frames or containing mutated vpr genes, base changes were made by site-directed mutagenesis (Quikchange II XL, www.stratagene.com) of subcloned fragments and cloning the mutagenized fragments back into DHIV. All mutations were sequenced to verify accurate mutagenesis, and Western blotting of lysates from DHIV-infected CD4pos Jurkat E6-1 cells was used to verify correct protein expression (Figure S8). DHIV-ΔVpu was verified to lack Vpu activity by its inability to down modulate CD4 molecules on primary T-cells (data not shown).
The FG12 vector system was graciously provided by Dr. Dong Sung An (University of California, Los Angles, CA). The DCAF1_3590 target sequences have been reported previously [42]. The scrambled sequence was 5′ GCATATCCACCGTGAGTGT 3′. All target sequences were obtained as olignucleotides (IDT, www.idtdna.com), annealed, and inserted downstream of the H1 RNA polymerase III promotor as described [59]. Western blots of lysates of primary activated T-cells transduced with the DCAF_3590 sequence produce less DCAF1 (Figure S9). pPR-Vip, the lentivirus vector that expressed HIV-1 Vpr and GFP, was generated as described previously [30].
Vesicular stomatitis virus (VSV)-G protein pseudotyped FG12, pPR-VIP and DHIV vectors were produced as previously described [36]. Viral vectors were titered by spin-inoculation at 1200× g for two hr at 20°C of dilutions of viral supernatant onto the Jurkat E6-1 cell line and detection of either intracellular HIV-1 p24 antigen (DHIV) or GFP expressing cells (FG12) by flow cytometry and the calculation of titers as described [36].
Primary T-cells were activated using anti-CD3/anti-CD28 mAb coupled to magnetic beads for 48 hr for DHIV and pPR-VIP or 24 hr for the FG12 vector. Infection/transduction was done by spin-inoculated as described [60] at an MOI of five for DHIV, ten for the FG12 vector, and five for pPR-VIP. For HIV-1NL4/3 infection; an MOI of 0.01 was used. Following the infection, the cells were cultured in RPMI complete medium with 100 U/mL recombinant IL-2 (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, deposited by Dr. Maurice Gately, Hoffmann-La Roche Inc, Nutley, NJ; [61]). For experiments where cells were transduced with the FG12 vector and then infected with DHIV, CD4pos T-cells were activated with anti-CD3/anti-CD28 mAb-coated microbeads, transduced with the FG12 vector, cultured for an additional 48 hr, and then infected with DHIV.
Flow cytometry
Simultaneous detection of surface antigens and intracellular HIV-1 p24 antigen (Ag) was done as previously described [24]. Infected cells were designated by the absence of CD4 and the presence of HIV-1 p24 Ag (Figure S1). For analysis of cell cycle profiles, cells were stained for the appropriate surface markers, washed with FACS buffer (PBS containing 0.1% NaN3 and 2% FBS) fixed for 20 min on ice with 0.25% paraformaldehyde, and permeabilized for 20 min on ice with 0.1% Triton X-100 in PBS. Following a wash in FACS buffer, intracellular staining was performed for HIV-1 p24 Ag and washed a final time. DNA was stained using 1 µM TO-PRO-3 (Invitrogen) in FACS buffer supplemented with 11.25 Kunitz units/mL RNAse (Sigma) and promptly analyzed on an LSR II flow cytometer (BD).
Purification of infected cells for cytotoxicity assays and real-time RT-PCR
Forty-eight hrs after infection culture, CD4neg p24pos cells were purified by removing CD4pos p24neg cells with anti-CD4 mAb coated magnetic beads (Dynal). To eliminate dead cells the Dead Cell removal kit from Miltenyi was used (http://www.miltenyibiotech.com/). Purity of infected cells was routinely greater than 95% as determined by flow cytometry.
Real-time RT-PCR
Primer pairs for detection of ULBP-1, ULBP-2, ULBP-3, MIC-A and MIC-B were obtained from previous studies described in [62]. The primer pair used for GAPDH was 5′ GCACCGTCAAGGCTGAGAAC 3′ (sense) and 5′ GGATCTCGCTCCTGGAAGATG 3′ (antisense). Total RNA was isolated using the RNAqueous Kit (Ambion, www.ambion.com) and treated with DNAse (TURBO DNA-Free, Ambion). Reverse-transcription and real-time RT-PCR reactions were carried out using the Superscript III Platinum Two-Step qRT-PCR kit using Sybr Green chemistry (Invitrogen) according to the manufacturer's protocol. The cDNA was amplified in triplicate with the ABI 7500 Real-Time PCR System (Applied Biosystems, www.appliedbiosystems.com) with indicated primer pairs for 40 cycles at 95°C for 15 sec and 60°C for one min. Analysis was done by using the ΔCT method for relative quantification as previously described [62] except that GAPDH was used as a reference. Similar amplification efficiencies for NKG2D ligand and GAPDH were demonstrated by analyzing serial cDNA dilutions with values of the slope of log cDNA amount vs. ΔCT of <0.1. Threshold cycles (CT) for GAPDH (reference) and NKG2D ligands (sample) were determined in triplicate. We defined the values obtained for uninfected cells as standard values and determined the relative increase (rI) in copy numbers in relation to these standard values according to the formula: rI = 2¯−[(CT Sample−CT Reference)−(CT Standard Sample−CT Standard Reference)].
Cytotoxicity assay
The cytotoxicity of HIV infected cells by autologous NK was measured using the 51Cr release assay. The cytotoxicity assay was done according to the methods previously described in [24].
Supporting Information
Figure S1.
Gating strategy used for detection of NKG2D ligands on infected cells. Infected primary T-cell blasts and uninfected CD4pos T-cells were surface stained with anti-CD4 Ab. All cells were stained intracellularly for HIV-1 p24 antigen (Ag). Cells were then incubated in the presence of Aquadead stain kit (Invitrogen) to distinguish viable and non-viable cells. Throughout the study NKG2D ligands were evaluated on either 104 viable uninfected (CD4pos HIV-1 p24 Agneg cells) or 104 viable infected cells (CD4neg HIV-1 p24 Agpos). FSC = forward scatter, SSC = side scatter. Gates in red indicate selection process for infected and uninfected cells.
Figure S2.
NKG2D ligands are not expressed on CD4pos T-cells infected with ΔVpr HIV-1. Infected primary T-cell blasts and uninfected CD4pos T-cells were surface stained with a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 antigen (Ag). Two-dimensional plots were derived following acquisition on a flow cytometer of 104 viable cells. Markers in dot plots were positioned based on the staining controls. The figure is representative data from three separate experiments.
Figure S3.
Effect of point mutations in postions 65 and 80 of Vpr on the cell cylcle of HIV-infected cells. Infected primary T-cell blasts and uninfected CD4pos T-cells were stained with TO-PRO-3 in order to obtain the (G2+M)/G1 ratio.
Figure S4.
Expression of DCAF1 is not affected by HIV-1 Vpr. The HeLa cell line was either treated with 10 µM aphidocolin (Aph), or infected with VSV-G pseudotyped HIV-1 with wild-type Vpr (Vpr) or HIV with Q65R and R80A mutations in Vpr (Vpr QR). Following treatment/infection cells lysates were made and western blotted. Western blots were probed with DCAF1 specific antibody.
Figure S5.
Inhibition of ATR activity relieves Vpr-induced G2 arrest. Primary CD4pos T-cell blasts were exposed to 4 mM of the ATR inhibitor, caffeine (B and D) or vehicle (A or C) and either infected with HIV-1 (C and D) or left uninfected (A and B). Forty-eight hrs. following exposure to caffeine and HIV-1 infection the cell cycle profile of the uninfected and infected cells were detected by TO-PRO-3 staining.
Figure S6.
Inhibition of ATM activity reduces NKG2D ligand expression on primary CD4pos T-cells treated with aphidocolin. Primary CD4pos T-cells were treated with 10 µM aphidicolin in the presence of 10 µM KU55933 (ATM-specific inhibitor) or 4 mM caffeine. As a negative control aphidicolin-treated cells were exposed to vehicles used to dissolve the inhibitor in solution. Following 48 h exposure to KU55933, caffeine or vehicle the cells were stained with a fusion protein of human NKG2D and the Fc portion of human IgG1 and fluorochrome conjugated-goat anti-human IgG Fc specific antibody (blue line) or secondary antibody alone [staining control (red line)]. The histograms are gated for 104 viable CD4pos cells. This is a representative of two experiments.
Figure S7.
Ability of NK cells to lyse autologous T-cells infected with HIV-1 lacking Vpr. Primary CD4pos T-cell blasts were infected with HIV-1 that were deficient in expression of Vpr (ΔVpr). As a control, the same cells were infected with wild-type (WT) HIV-1. Following infection the infected cells were isolated, labeled with 51Cr and mixed with autologous NK cells at 2.5∶1 (A and C) and 5∶1 (B and D) effector cell to target cell ratios. Prior to the lytic assay some of the NK cells were exposed to blocking antibodies to NKG2D (C and D). At the end of the incubation period culture fluids were harvested and analyzed for the presence of 51Cr. Percent specific lysis was determined as described in the Materials and Methods section. Each point designates a sample from each group. Bars represent the mean percent specific lysis. This supplemental figure is a dot plot representation of Figure 10.
Figure S8.
Expression of viral proteins by various HIV-1 mutants. HIV-1-infected CD4pos cells were lysed in cell lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.25% Na-deoxycholate, 1 mM EDTA. 1 mM PMSF, 1 mM Na3VO4, 0.1% SDS, and protease inhibitors), run on 15% SDS-PAGE gels, transferred to PVDF, and probed for the indicated proteins with specific antibodies. (A) Lysates from CD4pos cells infected with DHIVΔVpr, DHIVΔVif, DHIVΔVpr,ΔVif or DHIV containing Vpr with point mutations in specific residues. (B) Lyates from CD4pos T-cells infected with DHIVΔNef.
Figure S9.
Ability of shRNA expressing either scrambled or DCAF1 specific sequence to down modulate DCAF1 in transduced cells. Primary T-cell blasts were transduced with the shRNA with specific sequences, sorted by FACS to greater than 95% purity based on GFP expression, lysed in cell lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.25% Na-deoxycholate, 1 mM EDTA. 1 mM PMSF, 1 mM Na3VO4, 0.1% SDS, and protease inhibitors), run on 15% SDS-PAGE gels, transferred to PVDF, and probed for the either DCAF1 or β-actin proteins with specific antibodies.
The authors wish to thank Professor Alessandro Moretta (University of Genova, Genova, IT) for providing monoclonal antibodies used in the cytotoxicity assay and Dr. Dong Sung An (University of California, Los Angles, CA, USA) for providing the FG12 vector system. We would like to thank Dr. Linda Baum (Rush University Medical Center, Chicago, IL, USA) for her review and editing of the manuscript. We also thank Kelly Hudspeth and Bharatwaj Sowrirajan (Rush University Medical Center, Chicago, IL, USA) for their excellent technical assistance, and Dr. Alexander Steinle (University of Tübingen, DE) for helpful advice with the real-time RT-PCR.
Author Contributions
Conceived and designed the experiments: J. Ward, J. DeHart, E. Zimmerman, D. Mavilio, V. Planelles, E. Barker. Performed the experiments: J. Ward, Z. Davis, A. Bosque, E. Barker. Analyzed the data: J. Ward, Z. Davis, V. Planelles, E. Barker. Contributed reagents/materials/analysis tools: J. DeHart, E. Brunetta, D. Mavilio. Wrote the paper: J. Ward, Z. Davis, V. Planelles, E. Barker.
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- 53. Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, et al. (2003) Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates. Proc Natl Acad Sci U S A 100: 15011–15016.
- 54. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, et al. (2005) Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci U S A 102: 2886–2891.
- 55. Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, et al. (1999) The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10: 661–671.
- 56. Bonaparte MI, Barker E (2004) Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules. Blood 104: 2087–2094.
- 57. Bryceson YT, March ME, Ljunggren HG, Long EO (2006) Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood 107: 159–166.
- 58. Simm M, Shahabuddin M, Chao W, Allan JS, Volsky DJ (1995) Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes. J Virol 69: 4582–4586.
- 59. Qin XF, An DS, Chen IS, Baltimore D (2003) Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A 100: 183–188.
- 60. O'Doherty U, Swiggard WJ, Malim MH (2000) Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol 74: 10074–10080.
- 61. Lahm HW, Stein S (1985) Characterization of recombinant human interleukin-2 with micromethods. J Chromatogr 326: 357–361.
- 62. Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, et al. (2003) Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 102: 1389–1396.


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Senior Reporter
19 January 2018
Unclaimed cremated remains from parts of the Bukit Brown and Seh Ong cemeteries will be scattered at sea on April 26 this year, the Land Transport Authority (LTA) wrote in a series of final notices posted on national broadsheets.
As of 12 January, remains from about 3,400 out of 4,700 graves – exhumed from both cemeteries between January and December 2014 to make way for the construction of Lornie Highway – have not been claimed, said an LTA spokesman on Wednesday (17 January) in response to queries by Yahoo News Singapore.
These remains were not claimed by descendants or next-of-kin at the time of exhumation and during the storage period of three years, according to the notice published last Friday in The Straits Times and Lianhe Zaobao.
(A notice by the LTA published in Lianhe Zaobao on 12 January, 2018. SOURCE: Raymond Goh/Facebook)
Those who wish to claim the cremated remains of their ancestors have been asked to submit their claims by 12 April. They can contact the LTA at 63962500 or firstname.lastname@example.com for details on the submission of claims.
The LTA said it will arrange for a brief religious ceremony to offer prayers before scattering the ashes at sea.
Groups and individuals have called for alternative measures, such as building a memorial that houses the unclaimed cremated remains.
Chua Ai Lin, vice president of non-profit Singapore Heritage Society, called the suggestion "timely", and one that the group, together with members of the public and the Bukit Brown community, had proposed "a few years back".
As "some of the earliest graves exhumed to make way for the highway date back to as early as the 1830s, less than 20 years after the founding of Singapore by Raffles", a memorial would allow "current and future generations to pay their respects and remember the contributions of what remains of the earliest pioneer generation", explained Chua.
A memorial would play a big role in allowing the closure of a "chapter in (one's) family history", said Catherine Lim, a volunteer with All Things Bukit Brown, a community group that conducts regular guided walks for the public in the Bukit Brown area.
"The possibility that descendants may still come forth should not be discounted," added Lim.
Dr Terence Heng, lecturer in sociology at the University of Liverpool, who was part of a research team that documented the Bukit Brown cemetery, noted that building a memorial "would be an excellent gesture on the part of LTA".
"The memorial for Bukit Brown could be simple, placed near the recently relocated gates, listing the individuals who had 'given up' their space for the living," he added.
Plagued by delays
The construction of Lornie Highway, which was announced in September 2011, was delayed twice due to various reasons, including financial difficulties faced by its main contractor and a public exhumation exercise that completed a year later than projected.
First slated to be completed by 2016, and then end-2017, the development of the dual four-lane road also faced strong objections from heritage groups and members of the public.
Set to connect MacRitchie Viaduct to Adam Flyover, it affected parts of the Bukit Brown and Seh Ong cemeteries, which had been closed off to burial since the early 1970s and were areas deemed to have rich heritage value.
Lornie Highway will now be progressively opened from the third quarter of this year, starting with the southbound highway towards Adam Flyover, said the LTA in a press release issued in November last year.
The northbound highway towards MacRitchie Viaduct will open in the first quarter of 2019.
The road is expected to alleviate congestion along Lornie Road and the Pan-Island Expressway during peak hours.
Back in 2011, LTA said that "traffic demand along Lornie Road is expected to increase between 20 per cent and 30 per cent by 2020 and well beyond what the current Lornie Road will be able to handle".

subset: commoncrawl


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Information Architecture
The information architecture of a website determines more than just navigation, sections, headings. We recognise the value of a good "user experience", of finding data quickly and building information networks for your staff, members and visitors alike.
Katipo can help identify the specific needs of your user base and marry your website structure, data systems and functionality to their requirements. Our philosophy is that once the features and content are decided, the designer can then make a design which works in with the features and content, rather than trying to make the website features fit the design.
Card Storming
To help our clients organise their data, Katipo use Card-Storming, a cross between brainstorming and card sorting. Card storming involves you, the subject matter experts, arriving at a suitable site architecture with the help of your Katipo project manager, armed with marker pens and post-it notes. The site structure, features and functionality are refined over the course of a few meetings with your team and a few representative users if they're available.


text: The Hyundai Kona is a relative newcomer to the Hyundai SUV lineup, but it is already making a big impression all throughout the country with its efficiency and family-friendly features, making it an ideal choice for trips to The Villages to see grandparents or taking on the longer road trips ahead. Discover yours today here at Jenkins Hyundai of Ocala! We are located less than an hour outside of Dunnellon and we are ready to show you all that this brand-new mid-sized SUV can do for you!


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Peninsula Skin Cancer Centre has been owned by Australian Skin Cancer Clinics [Matrix] since 2015 however we have only just changed our signage in Frankston. We hope our bright new sign will help you find our same clinic and staff, with the large free car park.
New Fees from 1st June 2019
New Fees as of 1st of June 2019 FULL BODY SKIN CHECK: DVA patients are bulk billed. Concession patients will now be charged $60 for a 30 minute skin check, with a $38.20 Medicare Rebate, leaving an out of pocket cost of $21.80. Private patients will be charged $80 for a 30 minute skin check, with a $38.20 Medicare Rebate, leaving an out of pocket cost of $41.80. FULL 1 …Read More
369 melanomas
The total number of melanomas detected at Peninsula Skin Cancer Centre in 2019 was 369. This includes melanomas detected at Mornington and Frankston. Fortunately 98% of these melanomas were considered thin, less than 1mm deep, which means a wide excision is all that is required to treat that primary melanoma. All of these melanoma patients will require close surveillance for the next few years as they have an increased risk …Read More
Accredited Skin Cancer Doctors
This means that they have successfully completed a high level of training with the College and have proven skills in the diagnosis, treatment and management of skin cancer. Dr Mostafa Ziabari, Dr Jeremy Hay, Dr Sally Shaw and Dr Mary Tai Kie have all achieved Fellowship with the Skin Cancer College of Australasia. Dr Shamini Wiknesh and Dr Nadeeja Jayasinghe are currently working towards achieving fellowship. This means all our …Read More
Melanomas Detected at PSCC in 2017
Peninsula Skin Cancer Centre detected a record breaking 463 melanomas in 2017 in our two clinics. Over the last few years we have monitored the number of melanomas detected in our clinics and then analysed the number of melanomas for each age group, sex and the depth of melanomas. This valuable information helps us to monitor our performance at detecting melanomas on our patients and our ability to recognise melanoma …Read More
Sunscreen use can cut melanoma numbers
The results of a study by Brisbane's QIMR Berghofer Medical Research Institute has tried to quantify the benefits of applying sunscreen. Their study found that regular sunscreen use would reduce the incidence of melanoma in Australia by about 34 percent by 2031, which would translate into 28,000 fewer melanomas. QIMR Berghofer Medical Research Institute's professor David Whiteman hopes for a more realistic 5% increase in sunscreen use which would still reduce …Read More
Aerosol Sunscreen Warnings
The Australian Cancer Council have made an announcement stating they no longer recommend the use of spray-on and aerosol sunscreens. They have also withdrawn their own branded spray-on sunscreens from their online shop. The CEO of the Cancer Council, Professor Sancha Aranda, explained to reporters that Australians are not particularly good at the correct application of these types of sunscreens and that we should stick to using sunscreen creams and …Read More
National Skin Cancer Action Week
National Skin Cancer Action Week is the 19th to 25th of November in 2017. The theme this year is #SunSmartGeneration as the younger generations have been brought up with our Sunsmart message. This has led a decrease in the number of melanomas in this age group. The older generation however, continue to have an increase in the number of skin cancers, including melanoma. This age group have also shown a …Read More
70% of melanomas are new moles
A recent study published in the Journal of American Academy of Dermatology (A meta-analysis of nevus-associated melanoma: Prevalence and practical implications. Pampena R1, Kyrgidis A1, Lallas A2, Moscarella E1, Argenziano G3, Longo C4) showed that only 29.1% of more than 20,000 melanomas studied were associated with an existing mole with the remaining 70.9% melanomas arising de novo, that is from a new mole. With 70% of melanomas arising as a new mole, maybe we need to …Read More
Positive News For Melanoma Treatment
Researchers at Melanoma Institute Australia have released a report showing encouraging results for stage 3 melanoma patients, that is melanoma patients who have had melanoma spread to their lymph nodes. In the past, stage 3 melanoma treatments had very poor results in stopping the progression of melanoma metastasis, with 40-70% developing advanced and fatal melanoma. Researchers at Melanoma Institute Australia now have results from two trials that have encouraging results. …Read More


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Resources for Audiologists
Looking for resources? Below are some helpful links for audiologists:
The Ida Institute
This website offers free practical tools for audiologists focused on person-centered hearing care. It also includes counseling resources for adults and children on a variety of topics. The Ida Tools App is also available for Apple and Android.
Hearing Loss Association of America
This offers a resource to connect Deaf/HOH to area support group and educational meetings. This group is also very active in promoting hearing loops and hearing loop legislation across the country.
To support and empower members to provide the highest quality, life changing communication,
swallowing and hearing services to the people of the State of Indiana.


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Eric Wayne Thomas
December 19, 1983 - February 21, 2018
Eric Wayne Thomas born December 19, 1983 passed away February 21, 2018 at his residence at the age of 34. Eric worked construction as a Pile Driver and was of the Baptist Faith.
He leaves behind his beloved wife, Rebecca Voisin Thomas of Bogalusa, his father, Ben Albert Thomas of Bogalusa, a Sep-father, William david Young of Bogalusa, a son, Elijah Wayne Thomas , a step-son , Beau Anthony Voisin both of Bogalusa, 3 daughters, Eden Aline Thomas, Erica Leigh Thomas and Breanna LeAnn Thomas all of Bogalusa, a brother, Ben Albert Thomas II of Bogalusa, a nephew, Ben Albert Thomas III and other family members and friends.
He was preceded in death by grandfather, Leon Thomas and his mother, Linda Young.
Visitation will be held at Brown-McGehee Funeral Home on Tuesday February 27, 2018 beginning at 5:00 P.M. until 9:00 P.M. and will resume Wednesday February 28, 2018 after 8:00 A.M. until service time at 11:00 A.M. Bro. Jimmy Byrd will officiate the service with the interment to follow in the Spencer Cemetery


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Showing 1-5 of 5
By: N/A
$414.00 $372.60
Publisher: New York, McGraw-Hill Company, Inc.: 1959
Seller ID: 48129
Condition: Very Good-
All volumes are tight and clean! No noticed markings in any except for volume 15, which has some highlighting on a few pages. No other markings were found! Covers and slightly rubbed and edge worn. Overall excellent condition! ; The vast output of art criticism in our time has created invarious quarters the need for major historical synthesis covering the arts of all periods and countries - a kind of "corpus," both critical and documentary, which, within the framework of a single design and method, would seize the decisive factors and the most significant elements of that particular ... View more info
By: N/A
$7.00 $6.30
Publisher: Rochester, New York, Consumer Markets Division: 1978
Seller ID: 44677
Condition: Very Good with no Dust Jacket
Very little edge and corner wear. Pages tight and clean. No noticed markings. Very good condition. ; Pamphlet includes pictures and chart of all the parts of your camera. ; 3" x 4.5" View more info
By: N/A
$8.00 $7.20
Publisher: New York, The American Federation of Arts: 1958
Seller ID: 45369
Condition: Very Good+ with no Dust Jacket
Pages are tight and clean. Colors still vibrant. No noticed markings. Overall great condition! ; This book includes many photographs of paintings, sculptures, drawings and more by Harris K. Prior, Robert Goldwater, Leslie Cheek, Jr., and others. ; 6" x 9"; 177 pages View more info
By: Adams, Ansel
$28.00 $25.20
Publisher: Boston, New York Graphic Society: 1981
Seller ID: 56474
Condition: Very Good in Very Good Dust Jacket
Book and dust jacket are in great shape, very little wear, no noticed markings inside. ; ; The New Ansel Adams Photography Series Series; Vol. 2; 9.75" x 8"; 272 pages; 0821211315 View more info
By: Historical Society of Long Beach
$24.00 $21.60
Publisher: California, Historical Society of Long Beach: 1976
Seller ID: 30853
Condition: Very Good
Softcover book is in very good condition, with little wear and no noticed markings! ; ; 12" x 8"; 78 pages View more info


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Approved Used BMW M3 Cars in Blackheath
Finding the hunt for your perfect used BMW M3 car in Blackheath stressful? Venture slightly further to Woolwich, where you can find Stephen James Group. We are BMW specialists, so there is no dealership better to help you choose your next motor. BMW cars are celebrated for boasting an unbeatable drive and the BMW M3 is a prime example of this. If you want to see the used BMW M3 cars that we have in stock currently, you can find them all in our online showroom. Alternatively, you are welcome to come to our dealership near Blackheath. There are two Stephen James sites near Blackheath. It should only take twenty minutes to reach our MINI branch on Plumstead Road from Blackheath via the B210 or the A206. Or, you can find our approved used BMW cars by continuing past our MINI site to Duke of Wellington Avenue.
Every effort has been made to ensure the accuracy of the above vehicles information but errors may occur. Please check with a member of our team.
The representative finance examples shown above are for illustrative purposes only. Fees, rates and monthly payments may change subject to underwriting decision.
Representative APR
Representative APR 2.9%
*Fees are already accounted for within the payments displayed and are included within the total amount payable.
Finance is available to UK residents aged 18 years or older, subject to status. Terms & Conditions apply. Indemnities may be required. Other finance offers may be available but cannot be used in conjunction with this offer. We work with a number of carefully selected credit providers who may be able to offer you finance for your purchase, commission may be received. We are only able to offer finance products from these providers. Postal Address: Stephen James (Automotive) Ltd, Unit 1, Martinbridge Industrial Estate, 240-242 Lincoln Road, Enfield, Middlesex EN1 1SP. Find contact details here.
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Style, Fit & Opacity
- Fits true to size
- Slim-fitting through the shoulders
- High-cut neckline with ties, full-length sleeves
- Opaque
- Lightweight fabric
- Model is 180cm tall and a size 8 (UK), she is wearing a size 36 (FR) (UK08/IT40/US04)
Modist Memo
Animal print is a house signature for LAYEUR and the Evelyn shirt is a glamorous way to tap the season's animal print trend. Shaped for a relaxed fit with self-fastening neck ties that can be knotted into a bow, alongside long blouson sleeves and a button-down front, this modish offering feels utterly feminine. Emulate the label's modest take on occasion dressing by styling it with the coordinating jacket and trousers.
Styled here with: LAYEUR Trousers and Malone Souliers by Roy Luwolt Shoes
- Concealed button fastening through front with attached neck ties
- 100% viscose
- Dry clean
- Country of origin: Lithuania
Free returns within 14 daysFind out more
Find out the shipping options available for your destinationView
Personal Shopping
Need help with your order or styling advice?Contact us


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click here to download brochure
2 Chalets set in a glorious peaceful lakeside location nicely tucked in from a quiet tree lined Co. Cavan artery alongside Skeagh Lake. Chalet 1 - Sleep 8 and comprises - Ground Floor: Hallway, Living Room/Kitlchen, Bedroom, WC/Shower, First Floor: 3 Bedrooms all of which are Ensuite, Balcony. Chalet 2 - Sleep 6 and comprises - Ground Floor: Hallway, Living Room/Kitchen, WC/Shower, First Floor - 2 Bedrooms both of which are Ensuite, Balcony. Both chalets are spacious and tastefully deccorated - all bedrooms are ensuite. Also on site .... Sauna, Hot Tubs, Barbeque areas, Play areas, Lawns Lakeside activiites plus a host of country activities locally.


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Mom has done all of the work - now it's her time for a treat!
Soothing lotion - keeps Mommy soft and smelling sweet
Face mask - a little pampering for Mommy's face
Eye gels - get rid of those puffy, no sleep eyes
Dry shampoo - sometimes a shower isn't an option!
Hair band - makes for an easy care 'do
Lip balm - soft lips for kissing baby
Fuzzy socks - keep Mommy's toes warm during those late night feeds
Breast feeding scarf/bucket seat cover - go out on the town with baby in style!
Special cup - Mom needs hydration!


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Reply to the same customer questions with canned responses and save valuable time and effort. Save answers to common questions to shorten your response time and save keystrokes on repetitive typing. With canned responses, you type it once, save it and reuse it in chats and tickets whenever you need.
Canned responses are predefined messages displayed to the operators with the intent of answering repeated questions consistently. It helps the operators with swift assistance to the customers, operators can search for particular canned responses through the search box.
Welcome Messages
Here's how the Canned Responses appear to the operator in the Visitor Conversation Window:
Here's how to add canned responses in live chat:
Step 1: Navigate to "Settings"-----> "General Settings" -----> "Canned Responses."
Step 2: To add a new canned response click "Add New."
Step 3: Customize your canned response, you can also select responses from the library and add them to your account.
Here's how the library of pre-defined messages will appear, select an appropriate message and click "Save" to add it to your live chat.
To edit or delete canned responses, navigate to the specific response and click the edit or delete icon.
Related Articles:
Separate Chat Window
Visitor Chat Window Settings
Auto Chat Invitation


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Interleukin-10 | IL10
Alias: Interleukin-10, Cytokine synthesis inhibitory factor
IL-10 is an anti-inflammatory TH2 cytokine that has a critical role in limiting the immune response to pathogens to prevent host damage.
Though IL-10 is associated with TH2 response, it appears to be more relevant to TReg cell responses. IL-10 is known to be expressed by many adaptive immune cells including TH2, TH17, TReg, and B cells, as well as innate immune cells including dendritic cells (DC), macrophages, mast cells, natural killer (NK) cells, eosinophils, and neutrophils. As IL-10 in produced in several T helper populations, it is proposed that it provides a feedback loop to limit the effector functions of macrophages and DCs on T cells. Once expressed, IL-10 signals through the IL-10 receptor (IL-10R) to activate STAT3.
As IL-10 is a strong inhibitor of inflammation, it has become a viable biomarker for various diseases and conditions as well as a therapeutic molecule for certain conditions. In addition to elevated levels in parasitic infection, high expression levels of IL-10 are also found in retroviral infections inducing immunodeficiency. The immunosuppressive properties of IL-10 suggest a possible clinical use of IL-10 in suppressing rejections of grafts after organ transplantations.
IL-10 is capable of inhibiting synthesis of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα and GM-CSF made by cells such as macrophages and regulatory T-cells. It also displays a potent ability to suppress the antigen-presentation capacity of antigen presenting cells. However, it is also stimulatory towards certain T cells and mast cells and stimulates B cell maturation and antibody production.
Knockout studies suggested the function of Interleukin-10 / IL-10 as an essential immunoregulator in the intestinal tract.
Patients with Crohn's disease react favorably towards treatment with bacteria producing recombinant interleukin-10, showing the importance of interleukin-10 for counteracting excessive immunity in the human body.
The anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats.
This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.
Molecules. 2018 May 31;23(6). pii: E1325. doi: 10.3390/molecules23061325.
Study on the Structure of Ginseng Glycopeptides with Anti-Inflammatory and Analgesic Activity.
Luo H1, Zhu D2, Wang Y3, Chen Y4, Jiang R5,6, Yu P7, Qiu Z8.
Panax ginseng is well known for its medicinal functions. As a class of important compound of ginseng, ginsenoside is widely studied around the world. In addition, ginseng glycopeptides also showed good biological activity, but researches in this field are rarely reported. In this study, ginseng glycopeptides (Gg) were first prepared from Panax ginseng by reflux extracted with 85% ethanol and the following purification with Sephadex G-15 column. Then, the inflammatory pain models induced by carrageenan and the rat pain models induced by Faure Marin were established for research on mechanism of analgesic activities. It is showed that Gg had an obvious inhibiting effect on inflammation and a significant reduction on the Malondialdehyde (MDA) of inflammatory foot tissue. And there were significant differences between moderate to high dose of Gg and model group in Interleukin 1β (IL-1β), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Tumor necrosis factor α (TNF-α) and Histamine. The two models can be preliminarily determined that the analgesic effect of Gg may be peripheral, which mechanism may be related to the dynamic balance between proinflammatory cytokines (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-2, IL-4, and Interleukin 10 (IL-10)). A series of methods were used to study Gg in physical-chemical properties and linking mode of glycoside. The high-resolution mass spectrometry was used for identification of the structure of Gg. Moreover, the structure of 20 major Gg were investigated and identified. The structural analysis of Gg was benefit for the next study on structure-activity relationship.
Interleukin-10 Therapy—Review of a New Approach
The therapeutic role of interleukin-10 after spinal cord injury
J Neurotrauma. 2013 Aug 1;30(15):1311-24. doi: 10.1089/neu.2012.2651. Epub 2013 Jul 18.
Thompson CD1, Zurko JC, Hanna BF, Hellenbrand DJ, Hanna A.
1Department of Neurological Surgery, University of Wisconsin , Madison, Wisconsin, USA.
Spinal cord injury (SCI) is a devastating condition affecting 270,000 people in the United States. A potential treatment for decreasing the secondary inflammation, excitotoxic damage, and neuronal apoptosis associated with SCI, is the anti-inflammatory cytokine interleukin-10. The best characterized effects of IL-10 are anti-inflammatory-it downregulates pro-inflammatory species interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α, interferon-γ, matrix metalloproteinase-9, nitric oxide synthase, myeloperoxidase, and reactive oxygen species. Pro-apoptotic factors cytochrome c, caspase 3, and Bax are downregulated by IL-10, whereas anti-apoptotic factors B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X, B-cell lymphoma-extra large (Bcl-xl) are upregulated by IL-10.
IL-10 also provides trophic support to neurons through the IL-10 receptor. Increased tissue sparing, functional recovery, and neuroprotection are seen with an immediate post-SCI systemic administration of IL-10. Treatment of SCI with IL-10 has been used successfully in combination with Schwann cell and olfactory glial cell grafts, as well as methylprednisolone. Minocycline, tetramethylpyrazine, and hyperbaric oxygen treatment all increase IL-10 levels in a SCI models and result in increased tissue sparing and functional recovery. A chronic systemic administration of IL-10 does not appear to be beneficial to SCI recovery and causes increased susceptibility to septicemia, pneumonia, and peripheral neuropathy. However, a localized upregulation of IL-10 has been shown to be beneficial and can be achieved by herpes simplex virus gene therapy, injection of poliovirus replicons, or surgical placement of a slow-release compound. IL-10 shows promise as a treatment for SCI, although research on local IL-10 delivery timeline and dosage needs to be expanded.


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IT IS 3D were invited again this year to deliver STEM related activities at the YoMo event in Shanghai in July this year.
The experience was amazing. IT IS 3D teamed up with local company MakerBaby to deliver robotics, 3D printing, 3D scanning and 3D creative drawing.
The response we had was fantastic. Parents accompanying their children waited patiently for their turn to get a hands-on experience of the technologies on offer.
Robotic programming was popular with youngsters using symbol card recognition for the younger audience, as well as advanced robot arms where students were programming pick and place applications, with sorting items into bins and writing messages, amongst the activities.
We also exhibited dancing robots to encourage fun and fitness; this captivated everyone – from grown-ups to the youngest visitors to our booth, many of whom danced along with the robots.
It seems that an appetite for knowledge on new technologies is very much at the forefront of parents' and students' agendas. Over 4 days, Yomo attracted over 9,000 visitors, all very keen to get the hands-on experience on offer. Apart from IT IS 3D, there were other organisations showcasing AR, VR, electronics and programming, plus gaming to name but a few.
IT IS 3D will be attending Yomo again in 2018 in Shanghai where apart from offering hands-on activities on new and disruptive technologies linked to STEM, will also be involved in a competition involving STEM activities for students from age 6 – 21.
PS YoMo (Young Mobile) is an event which aims to excite and involve young people in current and future technologies. YoMo is organised by GSMA, the global organisation representing the mobile telephony and data industry. GSMA holds three annual conferences, in Shanghai, Barcelona and California.


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Dr Rebecca Huntley is one of Australians foremost researchers on social trends. Rebecca has a background in publishing, academia and politics and holds degrees in law and film studies and a PhD in Gender Studies.
For nearly 9 years Rebecca was the Director of The Mind & Mood Report, Australia's longest running social trends report.She currently heads Vox Populi Research, as part of the CIRCA research group.
Rebecca is the author of numerous books including Still Lucky: why you should feel optimistic about Australia and its people (Penguin 2017) and Australia Fair: Listen to the Nation, the first Quarterly Essay for 2019. She also writes for The Guardian and co-presented the Guardian's podcast Common Ground with Lenore Taylor in 2017. Rebecca is a broadcaster with the ABC and presents The History Listen each week on RN. She was a presenter of RN Drive on a Friday.
In addition to books, Rebecca has written extensively for essay collections, magazines, newspapers and online publications. She was a feature writer for Australian Vogue and a columnist for BRW and writes a column twice a month for ABC Life.
Rebecca co-hosts with Sarah Macdonald a comedy storytelling night and podcast called The Full Catastrophe. The Full Catastrophe book will be published by Hardie Grant in early May 2019.
Rebecca is on the Artistic Advisory Board of The Bell Shakespeare Company, is an adjunct senior lecturer at the School of Social Sciences at The University of New South Wales, is on the board of The Whitlam Institute and is a registered Commonwealth marriage celebrant.
Rebecca is married and the mother of three girls and lives in Sydney.


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Interactive media
What is Interactive Media?
Interactive media is a term used to describe a range of tools used to respond to a users actions. It can involve using a combination of video, text, music, animation, quizzes or graphics to achieve a learning goal.
Can you show me an example?
How long does it take?
The interactive learning asset creation process can take anywhere from 1 month to 5 months.
Academic staff will be required to input the main content at the beginning and will be able to review content throughout the production cycle. The process breaks down as follows:
- Once you have an idea (content) that you want to present as interactive media, write a short outline explaining the idea, aims and logic of interaction. Send it (alternatively you can schedule a meeting) with the content (may not be the final version) to the Digital Learning Specialist. Please feel free to be as descriptive and creative as possible if you think it will help the designer understand your ideas, please enclose some examples or sketches.
- Once the content is reviewed and everything is clear, the Digital Learning Specialist will get back to you with suggestions and a draft time schedule. At this stage the aim is to agree upon an user experience, logic, interactions, format, visual style, layout, pace and any other queries related to the final product. Depending on workload and period of the year (term times) it could take from 1 to 4 weeks.
- Production stage – Once the idea and style are agreed on, the production cycle begins. It may involve a lot of graphic design, 3D modelling, animating, digital composing, editing, and other specific techniques depending on the content. Depending on length, complexity and workload it could take from 1 to 4 months.
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- Distribution – The Interactive asset is being properly rendered, exported and distributed in the appropriate format.


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Two girls. One Leo.
Identical twins, Maggie and Clara Ryder, both grew up with Leo Maddox, billionaire playboy, apparent alcoholic, and heir to his grandfather's world-famous Maddox hotel empire. Their roles were cemented long ago: Maggie, as his best friend and Clara as his childhood nemesis.
But when a simple twist of fate changes everything and both girls start to fall for the lean, mean, ridiculously sexy and seemingly spoiled Leo... which girl will win his heart?
Throw in a little mischief, drama, and one smoking-hot bartender and lines are sure to blur. But one thing's for certain, neither good-girl Maggie nor rebellious Clara will be satisfied until they each figure out where they belong.
*NEW ADULT ROMANCE. Standalone. Recommended for readers 17+
*LEO MADDOX ~the alternative version to this novel is now available.


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Destination Sydney Exhibition
Olsen Irwin is thrilled to announce that two of our artists have been selected to be featured in the Destination Sydney Exhibition.
Destination Sydney is a multi-venue exhibition across the Manly Art Gallery, Mosman Art Gallery and S.H. Ervin Gallery designed to promote cultural tourism around Sydney.
Both John Olsen and Cressida Campbell have been selected to be part of a collaborative exhibition across three of Sydney's key public galleries.
Sydney has long been a point of focus and convergence for many artists born in other parts of Australia. The beauty of the city's bush harbour foreshores, the quality of light and palpable sense of energy and movement within the city has provided many artists an ongoing inspiration and a wealth of rich source materials from which to draw.
Each of the artists selected for this exhibition have been influenced by their experiences of living and working in Sydney that the city and its harbour have become central themes within their artistic production. Some of the artists selected were born in Sydney, but found that the city's pictorial and poetic possibilities inspired them only after a long stint travelling abroad.
Over 140 artworks have been lent for the exhibitions with many coming from private collections making this exhibition a must see.
Manly Art Gallery & Museum ~ LLOYD REES, BRETT WHITELEY, ELISABETH CUMMINGS
Mosman Art Gallery ~ JOHN OLSEN, KEVIN CONNOR, PETER KINGSTON
S.H. Ervin Gallery ~ MARGARET PRESTON, GRACE COSSINGTON SMITH, CRESSIDA CAMPBELL