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Cell Reports, Volume 2, Issue 6, 1736-1746, 20 December 2012
Induction of CD8+ Regulatory T Cells Protects Macaques against SIV Challenge
Wei Lu1, 2, , , Song Chen3, Chunhui Lai3, Weizhong Guo3, Linchun Fu3 and Jean-Marie Andrieu2, 4, ,
1 Unité Mixte Internationale 233, Institut de Recherche pour le Développement/Université Montpellier I, Montpellier 34394, France
2 Institut de Recherche sur les Vaccins et l'Immunothérapie des Cancers et du SIDA, Institut Necker, 156 rue de Vaugirard, Paris 75015, France
3 Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, PRC
4 Laboratoire d'Oncologie et Virologie Moléculaire, Centre Universitaire des Saints Pères, Université Paris Descartes, 45 rue des Saints Pères, Paris 75006, France
Corresponding author
Graphical Abstract
► Oral inactivated SIV adjuvanted by Lactobacillus plantarum induces tolerance to SIV ► MHC Ib/E-restricted CD8+ Tregs prevent SIV-harboring CD4+ T cell activation ► CD8+ Tregs suppress SIV replication in newly infected CD4+ T cells ex vivo ► CD8+ Tregs protect macaques from mucosal SIV challenge
- Efforts to develop a vaccine against HIV have so far met with limited success. Given that CD4+ T cell activation drives the initial burst of viral replication, we explored in macaques whether an oral vaccine comprised of Lactobacillus plantarum, a commensal bacterium that favors immune tolerance, and inactivated simian immunodeficiency virus mac239 (SIVmac239) would induce CD4+ T cell unresponsiveness/tolerance toward SIV antigens and thereby prevent the establishment of SIV infection. The tolerogenic vaccine induced MHC-Ib/E-restricted CD8+ regulatory T cells (Tregs) that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15 of 16 animals without inducing SIV-specific antibodies or cytotoxic T lymphocytes. Of 16 macaques that were intrarectally challenged with SIVmac239 or heterologous strain SIVB670, 15 were sterilely protected. In four macaques that were rechallenged intravenously, plasma SIV levels peaked slightly and then dropped to undetectable levels, although the animals subsequently harbored intracellular SIV DNA. Infusion of CD8 antibodies confirmed the role of CD8+ Tregs in preventing/suppressing SIV in vivo. These findings suggest a new avenue of research toward developing an HIV-1 vaccine.
Efficient vaccines against viral infections generally elicit virus-specific neutralizing antibodies and sometimes also cytotoxic T lymphocytes (CTLs) that prevent virus infection and/or eradicate the virus rapidly after it enters the body (Plotkin, 2010). So far, however, this process does not seem to work for HIV type 1 (HIV-1) infection, as shown by the failure to generate efficient long-lasting HIV-specific immunity via either neutralizing antibodies or CTLs induced by the vaccine prototypes that have been tested over the last 20 years (Fauci et al., 2008). Recently, the ALVAC-AIDSVAX vaccine trial showed that vaccinated individuals had a modest and short-term decreased incidence of HIV-1 acquisition (Rerks-Ngarm et al., 2009). However, more disturbing are the results of the STEP study, which showed an initial increase in virus acquisition in the group of vaccinated individuals compared with the placebo group (Buchbinder et al., 2008; Duerr et al., 2012). It is known that CD4+ T cell activation drives the initial burst of viral replication that leads to virus dissemination in vivo (Andrieu and Lu, 1995; Fauci, 1996). Therefore, the enhanced risk of infection observed in the STEP study could potentially be explained by the fact that the vaccine generated HIV-1-specific CD4+ T cells that on one hand may help CTLs to eradicate the virus, and on the other hand provide a substrate for efficient HIV-1 replication (Douek et al., 2002). Indeed, it has previously been shown that mucosal SIV transmission could be prevented in macaques by the local administration of an anti-inflammatory agent that prevented immune activation (Li et al., 2009). Against this background, we propose here that induction of HIV-1-specific tolerance might prevent the establishment of HIV-1 infection through suppression of CD4+ T cell activation.
To test this hypothesis, we used the model of the simian immunodeficiency virus (SIV) in macaques of Chinese origin, which best mimics HIV-1 infection in humans (Chen et al., 2011; Ling et al., 2002; Marcondes et al., 2006; Stahl-Hennig et al., 2007). We assessed whether an oral vaccine that favors immune tolerance and consists of Lactobacillus plantarum (LP) (Kleerebezem et al., 2003), a digestive tract commensal bacterium (Grangette et al., 2005; van Baarlen et al., 2009), and inactivated SIV would induce CD4+ T cell unresponsiveness/tolerance toward SIV antigens, which would in turn inhibit viral replication in vivo.
Induction of SIV-Specific Tolerance by an Oral Vaccine
In the first set of experiments, eight Chinese macaques were immunized via the intragastric route with aldrithiol-2 (AT-2)/heat-inactivated SIVmac239 (iSIV) (Rossio et al., 1998) formulated with LP, whereas control animals received LP only (four macaques) or iSIV only (four macaques). No difference was observed in the distribution of the classical major histocompatibility complex 1 (MHC-1) genotypes of the 16 animals (Document S1. Tables S1–S3). No side effect was observed after the vaccine was administered.
Because tolerance generally includes antigen-specific unresponsiveness of both the humoral and cellular arms of immunity, we looked first at SIV antibodies. We were unable to detect SIV-specific antibodies in the LP/iSIV-immunized monkeys, whereas we detected low titers of SIV immunoglobulin M (IgM) and IgG antibodies in the plasma of iSIV-treated controls (Figures 1A and 1B). Similarly, SIV IgA antibodies were not detected in the plasma and rectum washings of LP/iSIV-immunized monkeys or iSIV-treated controls, whereas they were detected in SIV-infected monkeys (Figures 1C and 1D). Due to the absence of reagents, SIV secretory-IgA antibodies were not checked. We then tested for SIV-specific cellular responses. Two months after immunization, SIV Gag-p27- and Gag-peptide-specific CD4+ T cell proliferation was not detected in any of the eight iSIV/LP-immunized animals, whereas it was present in the four iSIV-treated controls (p < 0.01). In contrast, SIV Gag-specific CD8+T cell proliferation was observed in both the eight iSIV/LP-immunized monkeys and the four iSIV-treated controls (p > 0.05; Figure 1E and Figure S1). Interferon γ (IFN-γ)-secreting T cells (CTLs) were also not detected upon SIV Gag p27 stimulation in the eight iSIV/LP-immunized animals, but were found (although at a low frequency) in the four iSIV-treated controls (p < 0.01; Figure 1F). We also depleted CD4+CD25+Foxp3+ regulatory T cells (Tregs) from the peripheral blood mononuclear cells (PBMCs) of immunized macaques using an CD25 monoclonal antibody. This had no effect on p27-specific T cell unresponsiveness in vitro (Figure 1F). Furthermore, we showed that there was no specific interleukin 10 (IL-10) or transforming growth factor β (TGF-β) release upon stimulation with SIV Gag p27 or Gag peptides in either the iSIV/LP-immunized or control animals (data not shown).
Suppression of SIV-Specific CD4+T Cell Activation by Nonclassical MHC-Restricted CD8+T Cells
We next investigated whether this form of immune tolerance involves a suppression of activation of SIV-positive CD4+ T cells. Because infected CD4+ T cells present Gag antigen derived from entering virions, i.e., before viral integration and viral protein expression (Sacha et al., 2007a), we pulsed fresh PBMCs from iSIV/LP-immunized macaques and control animals with nonreplicative (AT-2-treated) SIVmac239. As expected (Vatakis et al., 2009), ∼5% of the CD4+ T cells of all of the monkeys were "infected" by AT-2-treated (killed) SIV, as evidenced by intracellular p27 staining (Figures 2A and 2B). We then stimulated PBMCs overnight with staphylococcal enterotoxin B (SEB) and CD3/CD28 antibodies in the presence or absence (after depletion) of autologous CD8+ T cells and assayed for p27+CD4+ T cells bearing the early activation/proliferation marker Ki-67. We found that the percentage of activated (Ki-67+) p27+CD4+ T cells was much lower in PBMCs from iSIV/LP-immunized macaques than in PBMCs from LP- or iSIV-treated animals (mean ± SD, 8.9% ± 2.7% versus 49.1% ± 9.1% or 53.4% ± 7.0%, p < 0.01). Moreover, in PBMCs from iSIV/LP-immunized macaques, the percentage of Ki-67+p27+CD4+ T cells was also much lower in the presence than in the absence of autologous CD8+ T cells (mean ± SD, 8.9% ± 2.7% versus 54.5% ± 6.3%, p < 0.01). By contrast, in PBMCs from LP- or iSIV-treated animals, the percentages of Ki-67+p27+CD4+ T cells remained high in either the presence or absence of autologous CD8+ T cells (49.1% ± 9.1% and 53.4% ± 7.0%, respectively, p > 0.05; or 52.9% ± 7.4% and 57.5% ± 8.4%, respectively, p > 0.05; Figures 2C and 2D). We also showed that iSIV/LP-induced CD8+ T cells operated through a nonclassical MHC-restricted mechanism, because they suppressed the activation of nonreplicative SIV-pulsed CD4+ T cells from other iSIV/LP-immunized monkeys as well as those from control animals (p > 0.05; Figure 2E). Moreover, because the depletion of CD25+ cells did not modify the suppressive activity of PBMCs on p27+CD4+T cell activation (Figure 2D), we concluded that Tregs had no role in this suppression process. Finally, we controlled for the absence of CD4+ T cell lysis (p > 0.05) using a highly sensitive cytotoxicity assay (Marchetti et al., 1996; Figure 2F). Together, these results support the conclusion that a noncytotoxic mechanism underlies the CD8+ T cell-mediated suppression.
Suppression of Virus Replication by MHC-Ib/E Restricted CD8+ T Cells
Having demonstrated that adding LP to iSIV turned a classical SIV-specific immune response into an SIV-specific, CD8+ T-dependent tolerance, including the suppression of SIV-positive CD4+ T cell activation, we further studied whether the SIV-specific CD8+ T cells would suppress the replication of SIV in freshly infected CD4+ T cells ex vivo. We purified CD8+ T cells from PBMCs from the iSIV/LP-immunized macaques and added them to autologous CD4+ T cells acutely infected with SIVmac239. They were then stimulated overnight with SEB and CD3/CD28 antibodies. Over a period of 5 days, the CD8+ T cells conferred a strong antiviral activity in the coculture (p < 0.001) (Figure 3A). If the CD8+T cells were added more than 48 hr poststimulation (i.e., at a time when SIV-specific CD4+ T cell activation was well established), they could no longer inhibit viral replication (p < 0.001) (Figure 3B). This strongly suggests that the antiviral effect of the CD8+ T cells was generated by suppression of SIV-specific CD4+ T cell activation. We also demonstrated that the CD8+ T cell-mediated antiviral activity required cell-to-cell contact (p < 0.001) (Figure 3C) and was a classical MHC-unrestricted activity (as was the case for suppression of CD4+ T cell activation), as shown by the strong inhibition of viral replication by CD8+ T cells on acutely infected CD4+ T cells from other immunized animals in the group or from control animals (p < 0.001) (Figure 3D).
Finally, we showed that the CD8+ T cell-mediated antiviral activity was blocked by an MHC-Ib/E antibody but not by an ia/ABC antibody, indicating a nonclassical MHC-Ib/E-restricted CD8+T cell activity (p < 0.001) (Figure 3E). Clearly, T cell receptor (TCR) expression by CD8+ T cells is essential for recognition of MHC-Ib/E-peptide complexes carried by target CD4+ T cells (Sarantopoulos et al., 2004; Van Kaer, 2010). By depleting cell subsets in vitro with antibody-conjugated magnetic microbeads, we showed that TCRγδ and Vβ8 were not involved in suppression of viral replication (p > 0.05) (Figure 3F), suggesting that TCRαβ plays a central role in the recognition of MHC-Ib/E-peptide on infected CD4+T cells. However, even though we tested for a large number of CD8+T-cell-associated CD antigens (by depleting CD8+ T cells with human antibodies cross-reacting with membrane CD antigens of nonhuman primates), we were not able to identify any CD antigen associated with MHC-Ib/E-restricted CD8+ T cell activity (p > 0.05) (Document S1. Tables S1–S3). Finally, in an additional experiment, we found lower peripheral blood myeloid dendritic cell (mDC) and plasmacytoid DC (pDC) counts in protected (iSIV/LP-immunized) animals as compared with infected control animals (p < 0.01 and < 0.05 for mDC and pDC, respectively) or naive animals (p < 0.001 for both mDC and pDC) (Figure S2).
Protection of Macaques from Homologous and Heterologous SIV Challenges
Having established that oral immunization with iSIV/LP induced an SIV-specific tolerance that suppressed viral replication ex vivo, and that both suppression of CD4+ T cell activation and inhibition of viral replication resulted from the activity of non-IFN-γ-secreting TCRαβ MHC-Ib/E-restricted CD8+ T cells, we sought to determine whether such a tolerogenic vaccine would protect macaques from mucosal SIV infection. To that end, we challenged the eight iSIV/LP-immunized and eight control animals intrarectally with a high dose (100,000 TCID50) of SIVmac239 3 months after immunization (Chen et al., 2011). Surprisingly, the eight immunized animals were fully protected without any detectable level of plasma SIV RNA or PBMC proviral DNA. By contrast, the eight control animals were all infected (p < 0.001) (Figure 4A).
We further wanted to observe whether the animals that were protected against intrarectal SIV challenge could also be protected against an intravenous challenge. Two months after the intrarectal challenge, four out of the eight monkeys were challenged via the intravenous route with 200 TCID50 of virus. All showed a slight peak of SIV replication (≤200 DNA copies/million PBMCs and 200 SIV copies/ml of plasma) at day 10 postchallenge. However, by day 30, plasma SIV RNA had become undetectable (≤10 copies/ml) and PBMC SIV DNA had decreased to ≤10 copies/million cells, showing the absence of virus replication in vivo. In contrast, two naive animals that received the same intravenous SIVmac239 challenge were fully infected (p < 0.001) (Figure 4B). The four remaining monkeys were intrarectally rechallenged (100,000 TCID50) and, as expected, remained fully protected (p < 0.001) (Figure 4C).
To test whether vaccinated monkeys would also be protected from a heterologous challenge, 8 months later we further challenged the four intrarectally rechallenged monkeys and the four intravenously rechallenged monkeys with 100,000 TCID50 of the antigenically distinct SIVB670 via the intrarectal route. The eight animals remained fully protected, as shown by their undetectable SIV B670 DNA and RNA levels, whereas two control naive animals were infected by the same SIV B670 challenge (p < 0.001). This demonstrated that the SIV-specific tolerogenic vaccine was cross-protective, presumably through preventing the activation of CD4+ T cells that were infected by another SIV strain (Figure 4D).
Longevity of Vaccine-Induced Protection
We further immunized a new group of 16 macaques (eight with iSIV/LP, four with iSIV, and four with LP) (Document S1. Tables S1–S3) to study the longevity of the protection. The ex vivo antiviral activity of cells from this group was followed regularly. We observed that seven of the eight animals that received iSIV/LP maintained a high ex vivo antiviral activity of ≥100-fold compared with controls for up to 420 days (p < 0.001) (Figure 5A). As expected, no antiviral activity was detected in cells from animals treated with iSIV or LP alone.
On day 420 postimmunization, all 16 animals were intrarectally challenged with 100,000 TCID50 of SIVmac239. Seven out of the eight iSIV/LP-immunized animals had a sterile immunity as indicated by the absence of any SIV RNA and DNA emergence in plasma, PBMCs (Figures 5B and 5C), rectal mucosa lymphocytes (measured from day 1 postchallenge), or pelvic lymph nodes (Figure S3). Interestingly, one iSIV/LP-immunized macaque as well as all four of the iSIV-treated and all four of the LP-treated monkeys became fully infected (p < 0.001; Figures 5B and 5C; Figure S3). Moreover, the evolution of the ex vivo antiviral activity of the eight vaccinated monkeys allowed us to predict, from day 360 postimmunization (i.e., 60 days before their challenge), which one of the eight immunized monkeys would not be protected (Figures 5A and 5B).
Role of CD8+ T Cells in Suppressing SIV Replication In Vivo
In order to confirm the role of CD8+ T cells in vivo, we temporarily depleted CD8+ T cells from peripheral blood and lymphoid organs in the eight already challenged and protected monkeys in the first group of animals. Three intravenous injections of a mouse-human chimeric monoclonal CD8 antibody, cMT-807 (Centocor), were administered over a period of 1 week at days 300, 304, and 307 postimmunization (Figure 6A), and viral RNA and DNA were monitored. In the four macaques that had been rechallenged by the intrarectal route, no viral RNA or DNA was detected, demonstrating again their sterile protection. Interestingly and by contrast, a strong viral replication was observed in the four animals that had previously been challenged intravenously and harbored proviral DNA. In these animals, at the nadir of CD8+ T cell depletion (day 15), virus replication peaked at 106 RNA copies/ml of plasma, and lymph node proviral loads reached 104 DNA copies/106 cells (p < 0.001). However, by days 60–90, as the four animals recovered baseline CD8+T cell concentrations, virus replication was again brought under control, as indicated by plasma SIV RNA and PBMCs and lymph node SIV DNA returning to baseline levels (p > 0.05) (Figures 6B and 6C). This confirmed the unique role of the vaccine-induced CD8+ T cells in controlling viral replication in intravenously SIV-challenged animals in which replication-competent virus remained latent, presumably in quiescent memory CD4+ T cells (Willerford et al., 1990). A similar state of latency has been observed in HIV-1 patients treated with highly active antiviral therapy (Finzi et al., 1997).
Role of CD8+ T Cells in Protecting Macaques against Intrarectal SIV Challenge
To further define the role of CD8+ T cells in protecting against mucosal (rectal) challenge, we redepleted CD8+ T cells in the four macaques (#5–8) that previously had been sterilely protected through three intrarectal challenges (performed 3, 5 and 13 months postimmunization). The CD8 antibody (cMT-807) was administered 36 months postimmunization over a period of 1 week (days 0, 4, and 7) to temporarily deplete the CD8+ T cells in the same manner as described above. However, in this set of experiments, 2 hr after the first administration of CD8 antibody, the four monkeys were challenged intrarectally with 100,000 TCID50 of SIV239. As before, the cMT-807 treatment dramatically reduced the percentage of CD8+ T cells in PBMCs (Figure 7A). Consistent with our previous observations regarding the role of CD8+ T cells, all of the animals became infected. Indeed, at the nadir of the CD8+T cell count, the plasma viral loads of the four animals peaked at ∼104−106 RNA copies/ml (Figure 7B) and ∼103−104 copies of SIV DNA per million PBMCs (Figure 7C). By weeks 4–7, when the CD8+ T cell concentrations in the four monkeys had recovered to almost normal levels, plasma SIV RNA dropped to almost undetectable levels (10–50 copies/ml). This experiment further demonstrated the central role of CD8+ T cells in preventing initial infection at the mucosal barrier in immunized animals. When the CD8+ T cells were depleted, the virus replicated freely in lymphoid organs, but as soon as the vaccine-induced CD8+ T cells recovered, they again strongly controlled viral replication. Consistent with the above observations regarding intravenous challenge, the recovered animals now contained SIV DNA in target cells (Figure 7C), but without obvious viral replication (Figure 7B).
In the SIV-macaque model, we have demonstrated that infection can be prevented by inducing immunological tolerance against the infecting agent. The administration of iSIVmac239 and LP, a nonpathogenic commensal bacterium of the digestive tract that is known to induce immunologic tolerance, stimulated the macaques to develop a thus far unrecognized type of SIV-specific tolerance. This tolerance was characterized by the suppression of SIV-specific antibody and CTL responses, and activation of a subset of CD8+ T cells that are SIV-specific, noncytolytic, and MHC-Ib/E restricted. These cells apparently have the ability to suppress CD4+ T cells activated by SIV and thereby prevent the establishment of productive SIV infection both in vivo and in vitro.
These suppressive noncytolytic MHC-Ib/E-restricted CD8+ T cells represent a class of CD8+ Tregs that has not been described previously in the context of any vaccination or in SIV or HIV infection. Thus far, the only Tregs that have been proposed to be associated with HIV infection are CD4+ Tregs (CD25+FoxP3+CD4+ T cells) that are known to be involved in some models of immune tolerance (Faria and Weiner, 2005); however, their role in HIV infection is not clearly understood. On the one hand, the expansion of CD4+ Tregs has been shown to be associated with the suppression of HIV-specific CD4+ T cell responses and disease progression (Aandahl et al., 2004; Nilsson et al., 2006; Weiss et al., 2004), whereas on the other hand, CD4+ Tregs have been associated with protection from productive infection, CD4+T cell activation, and disease progression in both humans (Card et al., 2009; Chase et al., 2008; Legrand et al., 2006) and nonhuman primates (Jacquelin et al., 2009; Kornfeld et al., 2005). In the study presented here, the ex vivo removal of CD25+FoxP3+CD4+ T cells by a CD25 antibody did not modify either the suppression of CD4+T cell activation or viral replication.
Except for the fact that they are noncytolytic, the CD8+ Tregs observed here resemble those that target and eliminate abnormally activated antigen-specific CD4+ T helper cells in the mouse model (Kim et al., 2010; Sarantopoulos et al., 2004), where the inhibitory interaction depends on recognition of surface Qa-1 (corresponding to MHC-Ib/E in macaques and to HLA-E in humans) expressed by aberrantly activated target cells (Kim et al., 2011; Nagarajan et al., 2012). Similar CD8+ Tregs have also been implicated in the control of autoimmune type 1 diabetes in humans (Jiang et al., 2010).
Because monkeys challenged via the intrarectal route were negative for both SIV RNA in plasma and SIV DNA in PBMCs, our results suggest that virus infection was arrested before nuclear integration occurred. In quiescent CD4+ T cells, virus penetration is followed within 2 hr by the presentation at the plasma membrane of Gag/Pol and other protein epitopes derived from incoming virions (Sacha et al., 2007a), whereas Env and Nef proteins need de novo synthesis (Sacha et al., 2007b). The subsequent phases of the infectious process, including reverse transcription and proviral DNA integration, develop inefficiently in quiescent CD4+ T cells but very efficiently in activated CD4+ T cells (Korin and Zack, 1999; Zack et al., 1990). This is in keeping with the notion that the early activation of a small founder population of infected CD4+ T cells at the portal of entry is required for the local expansion and establishment of systemic infection (Haase, 2011; Zhang et al., 2004). This early event potentially provides vaccine-induced CD8+ T cells the opportunity to arrest the infectious process before proviral DNA integration occurs. We observed that the withdrawal of vaccine-induced CD8+ T cells from PBMC cultures before CD4+ T cell activation allowed the CD4+ T cells to become activated and viral replication to proceed. Similarly, in vivo depletion of CD8+ T cells performed at the time of intrarectal challenge allowed the CD4+ T cells to be activated and the virus to replicate. Such replication, however, was brought under control as the CD8+ T cells recovered in lymphoid organs and the blood stream. We showed in cultures ex vivo that the inhibition of viral replication was directly attributable to this class of CD8+ Tregs. This is highly likely to be the case in vivo as well; however, direct evidence is lacking because we were restricted to depletion of the whole CD8+ T cell population rather than the Treg subset specifically.
So far, we have not identified any CD antigen specifically associated with the vaccine-induced CD8+ Tregs observed in this study. The phenotypic and molecular characteristics of these CD8+ Tregs therefore require further study. Further studies are also necessary to elucidate how peripheral DCs are downregulated in vaccinated monkeys, because previous publications have reported that DC expansion is downregulated in vivo by classical Tregs (Kim et al., 2007; Liu et al., 2009).
In intravenously challenged monkeys, the transient peak of viral replication followed by the residual presence of cellular SIV DNA at a low level suggested that infectious SIV particles that entered the body by this route encountered activated CD4+ T cells, probably in secondary lymphoid organs, that allowed the virus to complete the first cycles of replication. We assume, however, that any newly released virions then entered nonactivated (quiescent) CD4+ T cells that were prevented from becoming activated by the LP/iSIV-generated CD8+ Tregs. Given that in vitro, SIV antigen-specific CD8+ Tregs of immunized macaques were effective enough to inhibit the superantigen SEB and CD3/CD28 antibody activation of infected CD4+ T cells (Figures 2C–2E), it is likely that in vivo, the activation of SIV-infected CD4+ T cells by SIV-unrelated stimuli known to upregulate viral replication (Andrieu and Lu, 1995; Fauci, 1996) will also be inhibited.
The protective effect of this innovative immunization regimen against SIV challenge, together with the identification of a correlate of protection in vitro, is quite striking. Given that SIV and HIV require activated immune cells in which to replicate, this tolerogenic vaccine approach may offer an exciting new avenue in HIV vaccine research. Moreover, the de novo induction of this class of CD8+ Tregs could potentially be used therapeutically to suppress HIV replication and prevent disease progression in place of antiviral therapy. Furthermore, the tolerogenic vaccine approach described here could potentially be exploited in the management of a wide range of immune disorders and is likely to have profound implications across immunology generally.
Experimental Procedures
Colony-bred rhesus macaques (Macaca mulatta) of Chinese origin were housed at the Nonhuman Primate Laboratory of the Gaoyao Experimental Animal Center (Guangdong, PRC) in accordance with the National Institutes of Health's "Guide for the Care and Use of Laboratory Animals." All animals were in good health, 2–4 years old, and weighed 4–6 kg, and were seronegative for SIV, simian retrovirus, simian T cell lymphotropic virus 1, hepatitis B virus, and herpesvirus simiae (B virus).
Viral Preparation
SIV was produced in CEM174 cells inoculated with SIVmac239 (gift of P.A. Marx; Chen et al., 2011). The culture supernatants were collected at peak virus production and inactivated first with 250 μM AT-2 (Sigma-Aldrich, St. Louis) (Rossio et al., 1998) and then by heating at 56°C for 30 min. The inactivated virus was inoculated to CEM174 cells to verify the 100% inhibition of viral infectivity.
Bacterial Preparation
LP (ATCC8014) was cultured at 37°C in MRS medium with a rotation rate of 200 rpm. To obtain LP at the logarithmic (midlog) phase of bacterial culture, bacteria were cultured until they reached an optical density at 600 nm of 1.0 with a final LP concentration of ∼1010 cfu/ml (∼3.5 hr) (van Baarlen et al., 2009).
Oral Delivery of the Vaccine
In the first study, 16 monkeys that had fasted overnight were anesthetized with intramuscular tiletamine hydrochloride and zolazepam (0.7 mg/kg). Eight animals (iSIV/LP #1–8) were then intragastrically administered 30 ml of a preparation containing 4 × 107 copies/ml of iSIV and 3 × 109 cfu/ml of living LP in maltodextrin (20%) solution. Monkeys received 25 ml of the same preparation intragastrically every 30 min for 3 hr. The same protocol was performed over five consecutive days. Four control animals (LP #1–4) received living LP alone, and the remaining four (iSIV #1–4) received only iSIV according to the same protocol.
In the second study, 16 new animals were recruited on the same screening basis: eight animals (iSIV/LP #9–16) received the same dose of the same vaccine, four (LP #5–8) were administered living LP alone, and four (iSIV #5–8) received only iSIV.
Assay for Antibody Responses to SIV
Plasma SIV IgG, IgM, and IgA antibodies were titrated by an immunofluorescence antibody assay (IFA) (Tsai et al., 1993) using fluorescein isothiocyanate (FITC)-conjugated goal anti-macaque IgG (Sigma), IgM (ADI, San Antonio, TX, USA), or IgA (ADI) antibodies. The sensitivity of the IFA assay was a titer of 20 for IgG and a titer of 5 for IgM and IgA. When a plasma sample was below the assay sensitivity, a value of one was assigned. Mucosal secretions were collected by washing the rectum with PBS using a catheter for gastric instillation as described previously (Méderlé et al., 2003). Anti-SIV IgA titers in the rectum were measured by the IFA assay.
Flow Cytometry
Flow-cytometry analysis was carried out with FACScalibur (BD Biosciences, San Jose, CA, USA) using the following fluorescence-labeled monoclonal antibodies: CD3-FITC (clone SP34-2), CD3-PE-Cy7, CD4-PE or CD4-PerCP-Cy5 (clone MT477), CD8-PerCP (clone RPA-T8), and Ki67-PE (BD Biosciences). FITC-conjugated P27 monoclonal antibody (Fitzgerald, Concord, MA, USA) or biotin-conjugated P27 monoclonal antibody (Fitzgerald) coupled with APC-SAv (BD Biosciences) was used for the intracellular staining of SIV-infected cells. Phycoerythrin (PE)-conjugated monoclonal antibodies against TCRγδ (clone B1), Vβ8, and CD antigens CD7, CD16, CD28, CD62L, CD95, CD122, CD137, CD150, CD183, CD184, CD195, CD196, CD197, CD226, CD272, and CD305 were purchased from BD Biosciences; PE-conjugated monoclonal antibodies against CD antigens CD11a, CD25, CD27, CD39, CD101, CD129, CD215, CD277, and CD357 were purchased from BioLegend (San Diego, CA, USA); and PE-conjugated monoclonal antibodies against CD antigens CD127, CD247, and CD279 were purchased from eBioscience (San Diego, CA, USA).
SIV-Specific Cell Proliferation Assay
PBMCs were obtained from fresh blood by centrifugation through Ficoll-Hypaque (Sigma). The proliferation of SIV-specific CD4+ or CD8+ T cells was evaluated by flow cytometry using the carboxy-fluorescein diacetate, succinimidyl ester (CFSE) labeling assay (Molecular Probes, Eugene, OR, USA). CFSE-labeled cells were stimulated for 5 days with 10 μg/ml recombinant SIV core protein P27 (ImmunoDiagnostics, Woburn, MA, USA), 2 μg/ml SIV Gag 15-mer peptides (GLS, Shanghai, PRC), 109/ml AT-2-inactivated SIV, or medium alone.
SIV-Specific ELISpot Assay
IFN-γ and IL-10 ELISpot assays were carried out in uncultured PBMCs in the presence or absence of SIV P27 using a commercial kit (Cell Sciences, Canton, MA, USA). A TGF-β1 ELISpot kit was purchased from R&D Systems (Minneapolis). The data were read with an automated ELISpot reader (AID, GmbH, Straβberg, Germany). The number of SIV P27-specific spot-forming cells (SFCs) was calculated by subtracting the nonspecific SPCs in the presence of medium alone.
T Cell Suppression Assay
Fresh PBMCs, depleted (or not) with either CD8 or CD25 by magnetic-bead-conjugated CD8 or CD25 antibodies (Miltenyi Biotec, Auburn, CA, USA), were pulsed with nonreplicative (AT-2-treated) SIVmac239 (at a viral concentration of 1010/ml) for 2 hr at 37°C in a 5% CO2 incubator. Pulsed cells were treated overnight with SEB (2.5 μg/ml; Sigma) and CD3 (2.5 μg/ml)/CD28 (2.5 μg/ml) antibodies (BD Biosciences) in RPMI 1640 culture medium (Sigma) supplemented with 1% fetal calf serum (Sigma) and 2% BSA (Sigma). Simultaneous intracellular stainings of SIV P27 and Ki-67 were performed 48 hr after the nonreplicative SIV pulse in order to determine the percentage of T cell activation (Ki-67+) within infected (P27+) cell populations. The P27+ cells were visualized with a fluorescent confocal microscope (Leica TCS SP2; Leica Microsystems CMS GmbH, Mannheim, Germany) using FITC-conjugated P27 antibody (Fitzgerald) with cytoplasmic staining by Evans Blue (Sigma).
T Cell Cytotoxicity Assay
Both purified CD8+ T cells (effector cells) and purified CD4+T cells pulsed with 1010 AT-2-treated SIVmac239 (target cells) were labeled with 40 nM 3,3′dihexyloxacarbocyanine (DiOC6) (Marchetti et al., 1996) (Molecular Probes) for 10 min at 37°C. Target cells were labeled with PerCP-Cy5-conjugated anti-CD4 (BD Bioscience) for 20 min on ice. Effector cells were washed three times and then mixed with target cells in a U-bottomed, 96-well plate at different effector/target ratios (3:1, 1:1, and 0.3:1) in triplicate. K562 cells (target) with APC-conjugated anti-CD32 (BD Bioscience) and purified CD56+ (NK) cells (effector) from four healthy donors were included as an assay control. After 4 hr of incubation at 37°C in the presence of SEB and anti-CD3/anti-CD28, cells were harvested and analyzed by flow cytometry. Percent cytotoxicity was calculated as follows: 100 × (% of total apoptotic target cells – % of spontaneous apoptotic target cells)/(100 – % of spontaneous apoptotic target cells).
SIV Suppression Assay
Autologous CD4+ T cells from each animal were purified by magnetic positive labeling (MicroBeads; Miltenyi Biotec) and then acutely infected with SIVmac239 (10−3 multiplicity of infection) in the presence or absence of magnetically purified CD8+ T cells at a CD4/CD8 ratio of 1:3, and then stimulated with SEB and CD3/CD28 antibodies for 16 hr. After washing, the cells were cultured in quadruplicates in 96-well plates. Cultures were maintained in a final volume of 200 μl per well of RPMI 1640 medium containing 100 IU of human rIL2 (Roche Diagnostics GmbH, Mannheim, Germany) for 5 days. Viral loads were measured by a real-time RT-PCR in culture supernatants collected at day 5 (see below). Fold suppression was determined as the geometric means of viral concentration in the culture supernatants from the infected CD4+ target cells only divided by the geometric means of viral concentration in the supernatants from the mixed CD8+ and CD4+ T cells.
In some experiments, CD8+ and CD4+ T cells were cultured without cell-to-cell contact by means of a Multiwell Insert System (BD Biosciences; CD8 in the insert well and CD4 in the bottom well), CD4+ T cells were cocultured with allogenic CD8+ T cells in order to determine the correlation between viral suppression and MHC restriction, and CD8+ and CD4+ T cells were also cocultured in the presence of MHC-ABC (BioLegend) or MHC-E (Cell Science) antibodies to define the modes of MHC restriction. To define the subsets of CD8+ T cells associated with antiviral activity, CD8+ T cells were purified from PBMCs immediately after their depletion with PE-conjugate TCRγβ, Vβ8, or other CD antigen antibodies using anti-PE microbeads through an LD column (Miltenyi Biotec).
Viral Challenges
Three months after oral administration of the vaccine or the controls, the 16 animals of the first study were inoculated intrarectally with 2,500 MID100 (100,000 TCID50) of pathogenic SIVmac239. Two months later, four vaccinated monkeys were rechallenged via the intrarectal route (100,000 TCID50) and four others were intravenously rechallenged with 5 MID100 (200 TCID50) of SIVmac239. As controls, two monkeys received an intrarectal challenge and other two received an intravenous challenge. These infectious doses generally result in a systemic infection in 100% of Chinese rhesus macaques with a peak plasma viral load (107−109 vp/ml) by days 10–14.
Thirteen months after the intragastric immunization (i.e., 8 months after the second challenge), the eight animals received a third intrarectal challenge with 100,000 TCID50 of pathogenic SIVB670 (a distinct strain of SIV provided by F. Villinger, Emory University School of Medicine, Atlanta, GA, USA). SIVB670 was propagated in vitro using primary macaque PBMCs, and the first passages of the original viral stocks were used for challenge. Again, two additional untreated macaques (Mac-naive #5–6) received in parallel an intrarectal challenge of SIVB670 as control.
In the second study, the 16 animals were intrarectally challenged on day 420 postimmunization with 100,000 TCID50 of SIVmac239.
Viral Loads
Cell-associated SIV DNA or plasma/supernatant SIV RNA was quantified by a quantitative PCR or RT-PCR with primers (sense, 5′-GAGGAAAAGAAATTTGGAGCAGAA-3′; antisense, 5′-GCTTGATGGTCTCCCACACAA-3′) and probe (5′-FAM-AAAGTTGCACCCCCTATGACA TTAATC AGATGTTA-TAMRA-3′) for SIVmac239 and primers (sense, 5′-TTGAGCCCTGGG AGGTTCT-3′; antisense, 5′-GCCAAGTGCTGGTGAGAGTCT-3′) and probe (5′-FAM-AACACCCAGGCTCTACCTGCTAGTGCTG-TAMRA-3′) for SIVB670 using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The sensitivity of the quantitative PCR or RT-PCR was three and ten copies for the input SIV DNA and RNA samples, respectively.
Depletion of CD8+T Cells In Vivo
Macaques were first anesthetized and then given an intravenous injection of a chimeric CD8 monoclonal antibody (cMT-807; Centocor Research & Development, Malvern, PA, USA) at 5 mg/kg on days 0, 4, and 7 (Schmitz et al., 1999). Peripheral blood samples (5 ml) were taken from each animal at day 0 and at various time points after antibody injections.
Statistical Analysis
Unpaired data from different groups of animals, and paired data obtained before and after immunization were compared by using the t, Mann-Whitney, and Wilcoxon tests, respectively.
Colony-bred rhesus macaques (Macaca mulatta) of Chinese origin were housed at the non-human primate laboratory of Gaoyao Experimental Animal Center (Guangdong, China) in accordance with the regulations of the National Institutes of Health 'Guide for the Care and Use of Laboratory Animals'. All animals were in good health, 2-4 years old, weighed 4-6 kg and were seronegative for SIV, simian retrovirus, simian T cells lymphotropic virus 1, hepatitis B virus, and Herpesvirus simiae (B virus).
MHC Class I Typing
Rhesus macaque MHC class I alleles were genotyped in PBMC using sequence-specific primers PCR assays for representative Mamu-A (senses/antisenses, 5′-GACAGCGACGCCGCGAGC CAA-3′/5′-GCTGCAGCGTCTCCTTCCCC-3′ for A01; 5′-TGTAAAACGACGGCCAGTGTG GGTGGAGCAGGAGGGTCC-3′/5′-CAGGAAACAGCTATGACCCAGCACCTCAGGGTG GCCTCT-3′ for A02; 5′-TGTAAAACGACGGCCAGTCCATGAGCTATTTCTACACCTA-3′/5′-CAGGAAACAGCTATGACCGGTAGGTTCTGTGCTGCTC-3′ for A04; 5′-TGTAAA ACGACGGCCAGTCCTTGAGGTATTTCTACACCG-3′/5′-CAGGAAACAGCTATGACC GCAGCCATGTCCGCTGCC-3′ for A08; 5′-TGTAAAACGACGGCCAGTGGGAGCCCC GCTTCTTCA-3′/5′-CAGGAAACAGCTATGACCTTCTGAGCCGCCATGTCCC-3′ for A11; 5′-TGTAAAACGACGGCCAGTATGAGGTATTTCTACACCTCCA-3/5′-CAGGAAACAGCT ATGACCCCAGGTAGGCTCTCATCCT for A13; 5′-TGTAAAACGACGGCCAGTTCCATG AGGTATTTCTACACCA-3′/5′-CAGGAAACAGCTATGACCCCAGGTAGGCTCTCATCCT-3′ for NA4; and 5′-TGTAAAACGACGGCCAGTATGAGGTATTTCTACACCTCCA-3′/5′- CAGGAAACAGCTATGACCCGCCACATCCGCCGCGAA-3′ for NA7) and Mamu-B (senses/antisenses, 5′-TGTAAAACGACGGCCAGTACCGGGAGACACGGAAGG-3′/5′-CAG GAAACAGCTATGACCAGCCACTCCACGCACCGG-3′ for B01; 5′-TGTAAAACGACG GCCAGTGGTATTTCAGCACCGCCGTA-3′/5′-CAGGAAACAGCTATGACCCACTTGCGC TGGGTGAACC-3′ for B07; 5′-CGTGAGGCGGAGCAGGTC-3′/5′-CCACAGCTCCGATGA ACACAG-3′ for B08; 5′-TGTAAAACGACGGCCAGTGCCGGGAACCCTGGTATC-3′/5′- CAGGAAACAGCTATGACCGAACCGCTCCGCATAACGGT-3′ for B12; 5′-TGTAAAACG ACGGCCAGTGCGACACGGAGAGCCAAGGA-3′/5′-CAGGAAACAGCTATGACCCCG CTCCGCATAACGGTTCC-3′ for B17; 5′-TGTAAAACGACGGCCAGTACTCCATGAGGT ATTTCACC-3′/5′-CAGGAAACAGCTATGACCCTTCTGCTCCGCCACACG-3′ for NB2; 5′-TGTAAAACGACGGCCAGTGCCGGGAACCCTGGTATG-3′/5′-CAGGAAACAGCTATG ACCGAACCGCTCCGCATAACGGT-3′ for NB4; and 5′-TGTAAAACGACGGCCAGTCCC CGCTTCATCTCCGTG-3′/5′-CAGGAAACAGCTATGACCTCGTAGGCGTCCTGCCGG-3′ for BN5) sequences (Loffredo et al., 2007; Mühl et al., 2002).
Viral Preparation
The SIV production was performed in CEM174 cells inoculated with SIVmac239 (gift of P.A. Marx). The culture supernatants were collected at pick viral production and inactivated first with 250 μM aldrithiol (AT)-2 (Sigma, St Louis, Missouri, USA), and then at 56°C for 30 min. The inactivated virus was inoculated to CEM174 cells to verify the 100% inhibition of viral infectivity.
Bacterial Preparation
Lactobacillus plantarum (LP) (ATCC8014) was cultured at 37°C in MRS medium with a rotation rate of 200 rpm. To obtain LP at the logarithmic (midlog) phase of bacterial culture, bacteria were cultured until reaching an optical density at 600 nm of 1.0 with a final LP concentration of around 1010 cfu/ml (about 3.5 hr).
Oral Delivery of the Vaccine
In the first study, 16 monkeys after having fasted overnight were anesthetized with intramuscular tiletamine hydrochloride and zolazepam (0.7 mg/kg). Eight animals (iSIV/LP#1-8) were then administrated intragastrically 30 ml of a preparation containing 4 × 107 copies/ml of iSIV and 3 × 109 cfu/ml of living LP in maltodextrin (20%) solution. Monkeys received intragastrically 25 ml of the same preparation each 30 min for 3 hr. The same protocol was performed over 5 consecutive days. Four control animals (LP#1-4) received living LP alone while the remaining 4 (iSIV#1-4) received only iSIV according to the same protocol.
In the second study, 16 new animals were recruited on the same screening basis; 8 (iSIV/LP#9-16) received the same dose of the same vaccine while 4 were administrated living LP alone (LP#5-8) and the remaining 4 (iSIV#5-8) only iSIV.
Flow Cytometry
Flow-cytometry analysis was carried out with FACScalibur (BD Biosciences, San Jose, California, USA) using fluorescence-labeled monoclonal antibodies: CD3-FITC (clone SP34-2), CD3-PE-Cy7, CD4-PE or CD4-PerCP-Cy5 (clone MT477), and CD8-PerCP (clone RPA-T8) (BD Biosciences). PE-conjugated monoclonal antibodies against TCRγδ (clone B1), Vβ8, and CD antigens CD7, CD16, CD28, CD62L, CD95, CD122, CD137, CD150, CD183, CD184, CD195, CD196, CD197, CD226, CD272, and CD305 were purchased from BD Biosciences; PE-conjugated monoclonal antibodies against CD antigens CD11a, CD25, CD27, CD39, CD101, CD129, CD215, CD277, and CD357 were purchased from BioLegend (San Diego, CA, USA); and PE-conjugated monoclonal antibodies against CD antigens CD127, CD247, and CD279 were purchased from eBioscience (San Diego, CA, USA). CD123-PE, CD11c-APC, HLA-DR-PeCy7, and a set of antibodies for defined lineages (B cells, T cells, and monocytes) (Lin-FITC) were purchased from BioLegend.
SIV-Specific Cell Proliferation Assay
PBMCs were obtained from fresh blood by centrifugation through Ficoll-Hypaque (Sigma). The proliferation of SIV-specific CD4+ or CD8+T cells was evaluated by flow cytometry using carboxy-fluorescein diacetate, succinimidyl ester (CFSE) labeling assay (Molecular Probes, Eugene, Oregon). CFSE-labeled cells were stimulated for 5 days with 10 μg/ml recombinant SIV core protein P27 (ImmunoDiagnostics, Wobun, MA, USA), 2 μg/ml SIV gag 15-mer peptides (GLS, Shanghai, China), 109/ml AT-2-inactivated SIV or medium alone.
Viral Loads
Mucosal viral load measurements were also performed in the second study. Rectum biopsies were performed in anesthetized macaques with the use of a fiber-endoscope (GIF-P20, Olympus Optical Co., Ltd., Japan). Isolation of intraepithelial and lamina propria lymphocytes from the macaque rectum were performed according to the standard protocol (Lefrançois, 1996). Cell-associated SIV DNA or SIV RNA was quantified by a quantitative PCR or RT-PCR with SIVmac239-specific primers (sense, 5′-GAGGAAAAGAAATTTGGAGCAGAA-3′; antisense, 5′-GCTTGATGGTCTCCCACACAA-3′) and probe (5′-FAM-AAAGTTGCACCCCCTATG ACATTAATCAGATGTTA-TAMRA-3′) using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, USA). The sensitivity of quantitative PCR or RT-PCR was 3 or 10 copies for the input SIV DNA or RNA samples, respectively.
Statistical Analysis
Impaired data between different groups of animals or paired data before and after immunization were compared by the t, the Mann-Whitney or the Wilcoxon test, respectively.
We thank W. Deng, V. Fabre Jagot, J. Yuan, L. Yu, J. Zheng, and Y. Zhou for technical assistance; Y. Yang for preparing the LP; S. Britton, J. Holmgren, and G. Karlsson Hedestam for critical readings of the manuscript; J.W. Almond for editing the manuscript; and P. Even and J.C. Weill for helpful discussions. This study was funded by a research contract between Biovaxim Ltd. (London, UK) and Université Paris Descartes (Paris, France), and the Institut de Recherche pour le Development (Marseille, France), and in particular was executed under a service contract between Institut de Recherche sur les Vaccins et l'Immunothérapie des Cancers et du SIDA (Institut Necker, Paris, France) and the Tropical Medicine Institute, Guangzhou University of Chinese Medicine (Guangzhou, PRC). S.C. was supported by a fellowship from Université Paris Descartes. W.L. and J.M.A. are shareholders of Biovaxim Ltd. and have received research support from the company. W.L. and J.M.A. were responsible for the overall study design, organization, data analyses, and writing of the paper. S.C., C.L., and W.G., assisted by L.F., participated in designing the study and performed experiments.
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Publication Information
Received: July 5, 2012
Revised: October 15, 2012
Accepted: November 21, 2012
Published: December 20, 2012


text:
Extravagant Gifts For Children Tres Chic: 13 Extravagant Gifts For Tots by Natasha Dantzig 11/22/11 1 Reactions 31 Shares Print 7 20 4 0 0 You've wrangled an invite to Beyoncé's baby shower, so now what? Whether you're actually shopping for a celebrity or just a friend who appreciates the finer things in life, finding a gift to satisfy tots at the top of the wealth stratosphere is no easy task. What do you get the kid that's got everything? These 13 luxurious gifts for kids fall way beyond the budgets of most people, but it sure is fun to look! Victorian Playhouse ($23,400) What's Your Reaction? 0 0 0 0 0 0 Once you get past the fact that this playhouse costs as much as some actual houses, it's hard to imagine that any tot wouldn't be completely dazzled by this Victorian Playhouse. Custom built, each playhouse can be outfitted with cable, running water, electricity, central air, or even a wireless communication system! 1 / 13 Baby Dior Exclusive Music Box ($156) What's Your Reaction? 0 0 0 0 0 0 The Baby Dior Exclusive Music Box plays the German nursery rhyme melody "Fuchs du hast die Gans Gestohlen," but aspiring fashionistas will be plenty soothed by the classic Dior box on its own. 2 / 13 Lionel Empire Builder Train Set ($2,800) What's Your Reaction? 0 0 0 0 0 0 Every lil boy wants a train set for Christmas. The Lionel Empire Builder Train Set is a real beauty that kids and collectors of all ages will treasure. Source: Flickr User jdpage 3 / 13 Little Queen of Love Youth Chair ($495) What's Your Reaction? 0 0 0 0 0 0 Lil princesses will rest their royal derrieres on the Little Queen of Love Youth Chair, fashioned in the opulent style of Louis XV. 4 / 13 Beige Fur Baby Blanket With Wool Lining ($545) What's Your Reaction? 0 0 0 0 0 0 Cuddle up with this Beige Fur Baby Blanket, but make sure you know a good fur cleaner: this rabbit fur version is as soft as it gets but requires a specialist. 6 / 13 Armani Junior Ivory Baby Bottles ($117) What's Your Reaction? 0 0 0 0 0 0 Not your average baby bottle, the Armani version features ivory lids. 6 / 13 Fendi Padded Zucca Snowsuit ($682) What's Your Reaction? 0 0 0 0 0 0 Tots will stay nice and toasty this Holiday season in this Fendi Padded Zucca Snowsuit with real fur trim, mittens, and booties. 7 / 13 Diamond Pacifier ($17,000) What's Your Reaction? 0 0 0 0 0 0 We told you about some of the best pacifiers for babies; this diamond pacifier, 14k gold and diamond studded, is the world's most luxurious. 8 / 13 Jeweled Classic Etch-A-Sketch ($1,500) What's Your Reaction? 0 0 0 0 0 0 A classic favorite, the Etch-A-Sketch gets a glittery makeover with this jeweled version. 9 / 13 Red Baron Pedal Plane ($548) What's Your Reaction? 0 0 0 0 0 0 Who doesn't like to play pilot? The Red Baron Pedal Plane is sure to top the list of tots with aeronautical aspirations. 11 / 13 Bonzini Babyfoot Barbie Foosball Table ($25,000) What's Your Reaction? 0 0 0 0 0 0 Barbie fans (and Charlie Sheen) will go nuts for this Barbie Foosball Table, a true collector's item. 11 / 13 Tiffany Piggy Bank ($1,350) What's Your Reaction? 0 0 0 0 0 0 It's doubtful that the tot who uses this sterling silver piggy bank really needs to be watching her pennies, but a little financial planning can't hurt. 12 / 13 Edible Gingerbread Playhouse by Dylan's Candy Bar ($15,000) What's Your Reaction? 0 0 0 0 0 0 This Edible Gingerbread Playhouse by Dylan's Candy Bar is one expensive treat! 13 / 13 7 20 4 0 0 Read More Extravagant GiftsKid ShoppingHoliday Gift GuideListHoliday Link Love Mom forced to stop feeding baby homemade formula — BabyCenter Blog The easiest ways to save on organic food — The Stir A look inside Fergie's second baby shower — Celebrity Baby Scoop Danielle Jonas debuts her baby bump — People Moms & Babies 7 light and fresh recipes to take you through Summer — FitPregnancy Does where you live affect your risk for PPD? — The Bump Where to find the best deals on school supplies — Real Simple Now this is how you announce a pregnancy! — HuffPost Parents
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I would like to enter my little dog, Afro Ken in your Halloween Contest. Ken is dressed up in a 'Jack in the Box' costume that I made for him. He got lots of treats for being such a good sport and I had so much fun with him that I made a "scary" video too. I hope you like it! :-)
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MOSCOW (Reuters) - The International Association of Athletics Federations (IAAF) is determined to crack down on drug-taking in the sport after two leading sprinters tested positive this month, the ruling body's vice-president Sebastian Coe said on Tuesday.
Coe was speaking in Moscow, the host of next month's world athletics championships, after American Tyson Gay, a former world 100 meters champion, and Jamaica's Asafa Powell, a former 100 meters world record holder, provided positive samples.
The two athletes have withdrawn from the Moscow championships.
"The right thing (to do) is being aggressive about our testing, is forever making sure our technology runs ahead of the competitors and our overall task is to make sure the sport is clean for clean athletes," Coe told Reuters.
"I think it would be unrealistic of me to say that we are ever going to reach a period where we will be entirely free from the scourge of drugs in sports," the former Olympic 1,500 meters champion added.
"But...we are taking this more seriously than we have ever taken it before."
The world championships will be the first major outdoor athletics event hosted by Moscow since the 1980 Olympics and it will be held at the same Luzhniki stadium which staged the opening ceremony of the summer Games more than three decades ago.
(Reporting Anastasia Gorelova, writing by Gabriela Baczynska, editing by Ed Osmond)


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Have been to both before and enjoyed reading the history of the attractions.
There are 2 sites dedicated to a couple of my favorite rides.
Dead Men... TELL NO TALES!
DoomBuggies > Explore the history and marvel at the mystery of Disney's Haunted Mansion attractions!
I hope you guys like them.
Have been to both before and enjoyed reading the history of the attractions.
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All Central America Travel Advice
February 25, 2014 3:57 pmWhether you're looking for tropical paradise, a nature-rich experience, or something just totally off the beaten path, there's a winning island for you. read more
February 14, 2014 2:05 pmWhether you're on the prowl for a perfect beach or a city bursting with culture, these cities and countries deliver big but cost little. read more
January 28, 2014 2:14 pmThis year's best new adventures include volcano trekking in Indonesia, tracking gorillas in the Congo, following a pilgrim trail through the Pyrenees, and seeking enlightenment in Japan's most remote alpine temples. read more
January 17, 2014 10:10 amWinter may be high season for the Caribbean, but that doesn't mean money-saving deals can't be found among its clear waters and sugar-soft sand. Head to a warmer clime (for a lot less dime) with one of these great deals. read more
January 15, 2014 1:06 pmEileen Ogintz takes a trip to Nicaragua, the country of lakes and volcanoes, beautiful beaches, and plenty of adventures. read more
January 2, 2014 9:25 am
November 15, 2013 10:07 amAt these dreamy digs on some of the world's best beaches, you can sink your toes into warm sand and find your ideal place in the sun. read more
October 11, 2013 2:39 pmIf you love exotic wildlife, ancient ruins, and otherworldly scenery, you might want to add these 10 foreign national parks to your bucket list. read more
August 30, 2013 10:03 amIt's best to be prepared, so here are some tips for Caribbean travel during the hurricane season. read more
June 11, 2013 2:22 pmSnorkeling, spider monkeys, and Mayan ruins! read more
May 31, 2012 10:32 amSee from the shore or hop aboard a boat! read more
May 30, 2012 9:22 amThis 107,000-acre reserve is home to exotic wildlife, Mayan artifacts, and cascading waterfalls. read more
May 10, 2012 9:18 amNeed to get away ... from other tourists? Book a stay on Royal Belize, a private island where anyone can stay—for a price. read more
March 22, 2012 9:30 amDiscover the laidback charms of West End Village on the tip of Roatan, Honduras. read more
March 21, 2012 8:43 amTobacco Caye may be tiny, but it's worth a visit—and it can accommodate most people's budgets. read more
March 8, 2012 9:15 amA treehouse for grownups! A free tubing adventure! Stays starting at just $40 a night! read more
February 29, 2012 9:15 amJacques Cousteau called this island "the most beautiful in the world." Who are we to argue? Find out where it is and how to get there! read more
January 26, 2012 9:10 amSalivate over these absolutely stunning properties in Europe, the Americas, Asia, the Middle East, and beyond. From Maui to Morocco and Italy to Ireland, these are the best of the best! read more
June 12, 2011 7:54 amSummer inspires vacations like no other season. All that demand can mean higher prices, but not if you know which surprising destinations are still affordable. Look to these five budget-friendly hot spots for a true summer bargains. read more
June 28, 2010 12:00 amYou'll have your choice of great deals and extra freebies (on a variety of islands) this summer in the Caribbean. read more
May 15, 2010 12:00 amSummer is one of the most popular times to travel. Luckily, the season does make some destinations off-peak, and smart vacationers can take advantage of lower rates around the world. read more
May 4, 2010 9:15 amFind out which destinations made the cut in TripAdvisor's annual list, voted on by travelers and readers like you. read more
April 27, 2010 12:00 amSpring and fall mean transitioning wardrobes—and repositioning ships. See several world regions on one of these sailings, at cheaper per-diem rates than "regular" cruises. read more
April 21, 2010 4:56 pmA few simple switches is all it takes to make a big difference to the environment—and your wallet. We asked Chris Lane, vice president of environmental affairs for Xanterra Parks, for advice, and also compiled some great tips from our readers. read more
April 6, 2010 10:13 amThe Deal Detective helps plan a trip to Belize on a budget, and adds a bit of adventure and eco-tourism without going overboard in cost. read more
April 1, 2010 4:30 pmApril 22 marks the 40th anniversary of Earth Day, and hotels in some of the country's most beautiful locations are celebrating with discounts and special activities. read more
March 27, 2010 12:00 amDreaming of a far-flung vacation? This year, those out-of-reach destinations may be surprisingly affordable. Read on to discover your budget-friendly vacation of a lifetime. read more
March 24, 2010 12:00 amThese trips aren't for everyone. But if crashing, roaring, flashing nature is up your alley, these destinations have dream vacation written all over them. read more
December 22, 2009 12:00 amIf you've ever thought you'd get seasick, bored, or not experience any culture on a cruise vacation, think again. Cruise Critic's Erica Silverstein debunks the most common excuses for avoiding a trip at sea. read more
August 19, 2009 1:34 pmThe Deal Detective helps plan a milestone birthday on a limited budget to places like Budapest and Costa Rica. read more
April 13, 2009 4:57 pmAdventure travel on a recession budget. What's not to love? read more
February 27, 2009 12:00 amMany airlines are launching new international service in 2009 to capitalize on new markets. Flying on introductory routes is a great way to save on overseas travel. read more
January 12, 2009 12:00 amEven in rough economic times, it's important to treat yourself to some relaxation. Preferably, it's nice to do so on a nice warm beach. I've found seven great, lesser-known beach destinations in the Caribbean and Atlantic that also go easy on your wallet. read more
December 23, 2008 12:00 amFrom the mountains of Rwanda to the hiking trails of Oregon, find out where we visited in 2008 and how we saved money doing it. read more
November 10, 2008 12:00 amWhile you're unlikely to get an all-expenses-paid trip unless you win a game show, you can still get portions your trip for free if you book certain promotions or take advantage of some alternative modes of travel. read more
April 20, 2007 12:49 pmSee fascinating wildlife, long-lost cities, and untouched beaches in these precious places. read more
April 4, 2007 8:53 amBelize is among the most expensive destinations in Central America, but it's also one of the most beautiful, accessible, and visitor friendly. And best of all, you can still visit on a reasonable budget. read more


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Luniac Style
Ever since I was a kid, I always told my dad that I wanted to make jewelry on a beach somewhere and earn my keep. Well, fast forward 25+ years later and here we are.
After having an on again off again love affair with making jewels, I made a clear decision to .. just do it. It wasn't the easiest decision, but it was the BEST one to make. There are days where you find yourself wondering if what you create will be well received, but I've realized that in the end, if you put love into ANYTHING, it will come back to you full force.
LuniacStyle was born about 8 years ago when some ex boyfriend told me I needed to "get a hobby". So I would work during the day at my 9-5, and work at night on my Luniac designs. Luniac was a word I made up meaning "crazy for the moon" (LUNA).
Luniac has come a long long way from that little apartment. I've moved a few times, worked at a few new jobs, got a new bf (who then became my huzzzzband) who didn't tell me I "needed" a hobby, but supports my dreams... and now next month I'm opening up a local shop/salon with my fabulous business partner, called LuniacGlamour! You're dreams never lead you astray. So put love into what you do, and make it your reality. LuniacStyle has been part of fashion shoots, fashion shows, Styleweek Providence, numerous blogs and articles, and the list goes on!
I'm so thrilled to have made my dreams a reality and hope it inspires everyone who has a tiny little dream. Hope you love my work!


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New york man convicted of hacking aT&T's servers
November 20, 2012 |
Stole E-mail Addresses and Personal Information Belonging to 120,000 Apple iPad 3G Subscribers; Disclosed Information to Internet Magazine
NEWARK, N.J. – A federal jury today convicted the head of a self-described "security research" hacking group of breaching AT&T's servers, stealing e-mail addresses and other personal information belonging to approximately 120,000 Apple iPad users, and disclosing that information to an Internet magazine, U.S. Attorney Paul J. Fishman announced.
Andrew Auernheimer, 27, of New York, was convicted of both counts of a Superseding Indictment: Conspiracy to access AT&T's servers without authorization and disclose that information to a reporter at Gawker magazine, and possession and transfer of means of identification for more than 120,000 iPad users. Auernheimer was tried before U.S. District Judge Susan D. Wigenton in Newark federal court. His co-conspirator, Daniel Spitler, 27, of San Francisco, Calif., previously pleaded guilty to the same charges and is awaiting sentencing.
According to documents filed in this case and the evidence at trial:
The iPad is a touch-screen tablet computer, developed and marketed by Apple Computers Inc., which allows users to, among other things, access the Internet and send and receive electronic mail. Since its introduction in January 2010, AT&T has provided iPad users with Internet connectivity via AT&T's 3G wireless network. During the registration process for subscribing to the network, a user is required to provide an e-mail address, billing address, and password.
Prior to mid-June 2010, AT&T automatically linked an iPad 3G user's e-mail address to the Integrated Circuit Card Identifier ("ICC-ID"), a number unique to the user's iPad, when he or she registered. Every time a user accessed the AT&T website, the ICC-ID was recognized and the e-mail address was automatically populated for faster, user-friendly access to the site. AT&T kept the ICC-IDs and associated e-mail addresses confidential.
At that time, when an iPad 3G communicated with AT&T's website, its ICC-ID was automatically displayed in the Universal Resource Locator, or "URL," of the AT&T website in plain text. Seeing this, and discovering that each ICC-ID was connected to an iPad 3G user e-mail address, hackers wrote a script termed the "iPad 3G Account Slurper" and deployed it against AT&T's servers.
The Account Slurper attacked AT&T's servers for several days in early June 2010, and was designed to harvest as many ICC-ID/e-mail address pairings as possible. It worked by mimicking the behavior of an iPad 3G so that AT&T's servers would be deceived into granting the Account Slurper access. Once deployed, the Account Slurper used a process known as a "brute force" against the servers, randomly guessing at ranges of ICC-IDs. An incorrect guess was met with no additional information, while a correct guess was rewarded with an ICC-ID/e-mail pairing for a specific, identifiable iPad 3G user.
From June 5, 2010, through June 9, 2010, the Account Slurper stole for its hacker-authors approximately 120,000 ICC-ID/e-mail address pairings for iPad 3G customers.
Immediately following the theft, the hacker-authors of the Account Slurper provided the stolen e-mail addresses and ICC-IDs to the website Gawker, which published the stolen information in redacted form, along with an article concerning the breach. The article indicated that the breach "exposed the most exclusive email list on the planet," and named a number of famous individuals whose emails had been compromised, including Diane Sawyer, Harvey Weinstein, New York Mayor Michael Bloomberg, and then-White House Chief of Staff Rahm Emanuel. The article also stated that iPad users could be vulnerable to spam marketing and malicious hacking. A group calling itself "Goatse Security" was identified as obtaining the subscriber data.
Goatse Security is a so-called "security research" group, comprised of Internet hackers, to which both Spitler and Auernheimer belonged.
During the data breach, Spitler and Auernheimer communicated with one another using Internet Relay Chat, an Internet instant messaging program. Those chats not only demonstrated that Spitler and Auernheimer were responsible for the data breach, but also that they conducted the breach to simultaneously damage AT&T and promote themselves and Goatse Security. As the data breach continued, so too did the discussions between Spitler, Auernheimer, and other Goatse Security members about the best way to take advantage of the breach and associated theft. On June 10, 2010, immediately after going public with the breach, Spitler and Auernheimer discussed destroying evidence of their crime.
Each count on which Auernheimer was convicted is punishable by a maximum potential penalty of five years in prison and a fine of $250,000.
U.S. Attorney Fishman credited special agents of the FBI, under the direction of Special Agent in Charge Michael B. Ward in Newark, with the investigation leading to the charges. He also thanked special agents of the FBI, under the direction of Special Agent in Charge Valerie Parlave in Little Rock, Ark., and the U.S. Attorney's Office for the Western District of Arkansas, under the direction of U.S. Attorney William Conner Eldridge.
The government is represented by Executive Assistant U.S. Attorney Michael Martinez and Assistant U.S. Attorney Zach Intrater of the Computer Hacking and Intellectual Property Section of the U.S. Attorney's Office Economic Crimes Unit.
Defense counsel: Tor Ekeland, Brooklyn, NY
Federal Emergency Management Agency (FEMA) announced that federal disaster aid has been made available to the State of New Jersey.
FEMA is now accepting applications from residents and business owners who sustained losses in Atlantic County, Cape May County, Essex County, Hudson County, Middlesex County, Monmouth County, Ocean County and Union County.
Register online at
www.disasterassistance.gov , by web-enabled mobile device atm.fema.gov or by calling 1-800-621-FEMA(3362) or 1-800-462-7585 (TTY) for the hearing and speech impaired. The toll-free telephone numbers will operate from 7 a.m. to 10 p.m. EDT seven days a week until further notice.


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Even the rain didn't stop these stars from shining on the Oscars red carpet at the 86th Academy Awards. click here
THIS WEEK'S HOTTEST PICS! click here
2014 Awards season fashion and nail trends click here
All the stunning red carpet looks from the British Film Industry's night of nights! click here
The hottest pics of the week starting February 24 click here


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On View:
Friday, May 14, 2010 to Sunday, January 9, 2011
Cooper-Hewitt, National Design Museum presents the fourth exhibition in the National Design Triennial series. Why Design Now? explores the work of designers addressing human and environmental problems across many fields of design, from architecture and product design to fashion, graphics, new media and landscape design. Organized by Cooper-Hewitt curators Ellen Lupton, Cara McCarty, Matilda McQuaid and Cynthia Smith, the Triennial will be global in reach for the first time, reflecting the connectedness of design practices and the need for international cooperation to solve the world's problems. Read the full press release.


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*Northeast storm lives up to its monster billing ("Snow totals surpass 2 feet, 650,000 homes and businesses without power as massive blizzard wallops Northeast.") *Obama will propose 1% raise for federal workforce in 2014 budget *Spike of Virginia redistricting plans shows House speaker's pragmatic streak *Clinton: America's Debt Problems 'Can't Be Solved' With Austerity *Jindal speaks at annual Cantor fundraiser; urges growth for GOP *Warner: Sequestration Would Be 'Worse Than You Can Imagine' *Warner: Defense industry shares blame for sequester *Virginia's hi-tech corridor nervous over sequestration *Mr. Cantor looks for middle ground *Virginia General Assembly: The score at halftime *Bolling to make "major announcement" March 14 *McDonnell, Senate Dem leaders exchange road notes *Politics over sense in I.D. measures ("The notion that this measure ensures the integrity of elections has little basis in reality.") *McDonnell sends school security bills to lawmakers *Hey Fairfax County, High School Seniors Can Handle 'Beloved,' And Learn About Racism and Sexism *Anti-uranium legislators make their case to McDonnell *Putney considering a re-election bid as a Republican *This time, Roanoke Valley got biggest snowfall totals
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BUSTED!: Female Pedo World Of Warcraft Player Arrested Upon Return To Texas
Remember the story about the 16-year old boy and 42-year old cougar hyena that met playing World of Warcraft and decided to bump warlocks in real life? Well the woman was arrested at the airport in Texas after returning from visiting the boy in Canada. And her nipples were still hard from the cold.
Donna Hawkins, a spokesperson for the Harris County District Attorney's Office, told CTV.ca that the two allegedly "engaged in a cybersexual relationship" for more than a year.
It is alleged that Price and the teenager "engaged in some sexual activity" before they were discovered, said Hawkins.Barrie police questioned Price, but found that she had not broken any law since the age of consent is 16.
In Texas, however, the age of consent is 17, and Harris County prosecutors charged Price despite the fact that the alleged offence did not occur locally, said Hawkins.
Price has been charged with two counts of online solicitation of a minor and one count of child enticement. None of the charges have been proven in court.
Interesting, but do you know what has been proven in court? My right to flex. Second amendment baby! *swish*
Texas woman who flew to meet Barrie teen, arrested [sympatico]
Thanks to Alioth, who's a level 80 Chris Hansen.
Leeeeeeeeeeroy Jenkins! I meant to post this story last month, but, I'm going to be perfectly honest with you, I got drunk and forgot about it. But now we know the results so I don't have to write about it twice, which works out for me because words can be tricky to type. An... / Continue →
A French gamer spent 7-months hunting down another Counter-Strike player so he could stab him in real life after the guy killed him during an in-game knife fight. Geez, somebody's high school guidance counselor didn't do their job! Julien Barreaux, 20, told police he wanted t... / Continue →
Note: Hit this pretty in pink button right HERE to see the entire graphic. This is an informational graphic with some facts and figures about World of Warcraft. But that isn't the whole thing so would you kindly (I OWN YOUR ASS!) click the link above to see it in its entirety... / Continue →


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In multiples of 20 these coins will be shipped in a plastic tube (not the original mint tube)
Make a solid investment in a beautiful piece of history
Minted by the Casa de Moneda de Mexico in limited quantity and issued annually since 1982, the 1oz Mexican Libertad Silver Coin is one of the most popular bullion coins in the world.
- • Guaranteed weight – 1 Troy ounce (31.1 g) of silver
- • Guaranteed purity – 99.9% pure silver coin
- • Sustainably high demand – low mintage & variable design
The obverse of the coin shows the coat of arms of Mexico surrounded by historical Mexican coats of arms. The reverse depicts a winged Victoria of Mexican Independence Victory Column in front of a landscape with the volcanoes Popocatépetl and Iztaccihuatl.
The Mexican Mint is the oldest mint in the Americas and was established in 1535. The interesting fact about this mint is that its silver eight-real coins and later on the silver peso became the basis of the modern national currencies of many countries in this part of the world, including the US dollar, Chinese yuan and Japanese yen. Another interesting fact related to this part of the world is that the widely used $ symbol used to represent the Spanish American Peso during 1770s, also known as Spanish dollar, in British North America, and was later on used as a model for the currency adopted by the United States in 1785.
In multiples of 20 these coins will be shipped in a plastic tube (not the original mint tube).


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Specialty Korean foundry earns essential certification to fabricate high-reliability ICs for demanding defense, aircraft and outer space applications
SEOUL, South Korea, July 25 (Whowired)--Dongbu HiTek today announced that it has been certified to implement high-reliability ICs per the rigorous MIL-PRF-38535 standard using its specialized mixed-signal, BCDMOS process at both the 0.18- and 0.35-micron nodes. Granted by the U.S. Defense Logistics Agency (DLA), this performance certification adds the specialty Korean foundry to a list of subcontractors eligible to produce ICs per the applicable MIL-PRF-38535J and MIL-STD-883H specifications.
With today's announcement, Dongbu HiTek's specialized mixed-signal, BCDMOS processing promises to become a preferred option for the Qualified Manufacturer's List (QML) of contractors developing products for DLA-managed programs. According to a company spokesperson, "We have already begun implementing mixed-signal ICs for a world leading fabless company specializing in the design of high-performance, high-reliability, radiation-hardened ICs for defense, aircraft and outer space applications. We continue to differentiate our foundry services by focusing on specialized processing that adds high-value to ICs demanding the highest levels of quality and reliability."
About MIL-PRF-38535J
This specification defines the general performance and verification requirements, as well as the quality assurance and reliability criteria, for the manufacture of semiconductor devices used in military and high-reliability applications. It provides device manufacturers with an acceptable established baseline to support Government applications and logistic programs. For QML devices the manufacturer is required to develop a program plan that meets or exceeds the performance of criteria detailed in appendices of this specification. Among those devices addressed in the appendices are those that must comply with MIL-STD-883 as well as those intended for space applications and those that require radiation hardening assurance (RHA).
About MIL-STD-883H
Establishing the uniform methods, controls, and procedures for testing microelectronic devices suitable for use within military and aerospace electronic systems, this specification includes basic environmental tests to determine resistance to deleterious effects of natural elements and conditions surrounding military and space operations; mechanical and electrical tests; workmanship and training procedures; and such other controls and constraints as have been deemed necessary to ensure a uniform level of quality and reliability suitable to the intended applications of those devices. The term "devices" used in this standard includes monolithic, multichip, film and hybrid microcircuits, microcircuit arrays, and the elements from which the circuits and arrays are formed.
About Dongbu HiTek
As the World Leader in Specialty Foundry, Dongbu HiTek Co., Ltd. specializes in delivering superb analog and mixed-signal processing technologies. The company adds high value to advanced ICs with a processing portfolio that encompasses Analog CMOS, BCDMOS, High Voltage CMOS, CMOS RF, CMOS Image Sensor (CIS), Display Driver IC (DDI), and NOR Flash technologies. With its best-in-class BCDMOS technology, the company enables power management chip designs that serve numerous applications ranging from home and handheld electronics to automotive and industrial systems. Currently processing technologies at nodes ranging from 0.35 microns to 90 nanometers, the company complements its world class foundry capabilities with top quality design support, prototype verification and packaging/module development. Dongbu HiTek publicly trades its stock under 000990 on the Korea Stock Exchange. For more information, visit www.dongbuhitek.com.
Dongbu HiTek HQ
Kevin Lee, +82.32.680.4052
Dongbu HiTek USA
Elizabeth Estrella-Basilio, +1-408-238-8822


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More copies of this ISBNThis title in other editionsThe Mapmaker's Wife: A True Tale of Love, Murder, and Survival in the Amazonby Robert Whitaker
Synopses & ReviewsPublisher Comments:A True Tale of Love, Murder, and Survival in the Amazon
The year is 1735. A decade-long expedition to South America is launched by a team of French scientists racing to measure the circumference of the earth and to reveal the mysteries of a little-known continent to a world hungry for discovery and knowledge. From this extraordinary journey arose an unlikely love between one scientist and a beautiful Peruvian noblewoman. Victims of a tangled web of international politics, Jean Godin and Isabel Gramesóns destiny would ultimately unfold in the Amazons unforgiving jungles, and it would be Isabels quest to reunite with Jean after a calamitous twenty-year separation that would capture the imagination of all of eighteenth-century Europe. A remarkable testament to human endurance, female resourcefulness, and enduring love, Isabel Gramesóns survival remains unprecedented in the annals of Amazon exploration. About the AuthorRobert Whitaker is a science journalist and the author of Mad In America. He has won the George Polk Award and was a finalist for the 1998 Pulitzer Prize for Public Service. He lives in Cambridge, Massachusetts.
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See the world through a superhero's eyes when you transform from playboy to Iron Man with our official Iron Man Goggles. One size fits most adults and make a cool alternative to masks and make up.
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About Us
The Neon Angels is a fashion design/ stylist/ PR duo living in Los Angeles, CA. Working globally, we have put in our time with some of the most prominent names in the industry. Setting the standard, and breaking the rules: We're here to put you on.Contact
email@example.com's be Friends
Tag Archives: Judith Bedard
Thief of Hearts: Judith Bedard x Soma magazine
A little editorial that I just fell in love with, Judith Bedard shot x Filippo Del Vita for the November edition of Soma. … Continue reading
Posted in Fashion, Photography & Editorial
Tagged Avant Garde, black, Editorial, Fashion, gold, Judith Bedard, Models, Photography, Soma, styling
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Case against man who texted photo of his tattooed penis dismissed
A man accused of texting an unsolicited picture of his tattooed penis to a married mother did not commit a crime Georgia's Supreme Court has ruled.
Fugitive banker wanted for fraud caught
A former Georgia banker who left a suicide note and vanished while under suspicion he embezzled $21 million has been captured 18 months after he vanished.
Ira Curry: Georgia woman named one of 2 Mega Millions winners
The winner, Ira Curry, played a combination of winning numbers that she said were based on family members' birth dates when she bought the ticket at an Atlanta newsstand.
Winning Mega Millions lottery tickets sold in California, Georgia
A store owner in Atlanta said she was thrilled to learn on Wednesday that her shop, and another in San Jose, California, had sold winning tickets for an estimated $636 million Mega Millions jackpot, the second biggest in U.S. lottery history.
Auburn football beats Georgia on epic Hail Mary
No. 7 Auburn was trailing No. 25 Georgia by one point with just 36 seconds left in Saturday's SEC West slugfest.
Police find body of 10-year-old girl in trash can in Lawrenceville, Ga.
Police made a gruesome discovery in a trash can in Lawrenceville, Ga.
Kidnapped teen Ayvani Hope Perez found alive, suspects arrested
Ayvani Hope Perez, the 14-year-old Georgia teen who was violently abducted during a home invasion, has been found alive, according to CNN.
Ayvani Hope Perez: Friends of kidnapped Georgia teen turn to social media
Just hours after 14-year-old Ayvani Hope Perez was taken from her Atlanta-area home, friends and community members have turned to Twitter and Facebook in an effort to spread the word and bring her to safety.
Shelter promised to rescue dogs, killed them instead: authorities
A Georgia animal shelter's "Lucky Dog" adoption program deceived pet owners by promising not to euthanize their dogs for a $100 fee, then killed them instead, authorities said.
After originally denied, dying teen Anthony Stokes will be placed on donor list
Anthony Stokes will now be put on the donor list after originally being denied due to "non-compliance," outraging locals.


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Laura Branigan
• Celebrity Birthdays, July 3
• Celebrity Birthdays, July 3
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Gloria / Living A Lie [Digital 45]
A Much, Much Greater Love
The Best Of Branigan
Over My Heart All Albums >>
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On: Memories |
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Solitare On: The Best Of Branigan |
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Shadow Of Love On: Touch |
Laura Ann Branigan (July 3, 1957 – August 26, 2004) was an American singer-songwriter and actress of Italian and Irish ancestry. She is best known in the United States for her 1982 Platinum-certified hit "Gloria" and in Europe for the number one single "Self Control". Branigan is also remembered for the Top 10 "Solitaire," and for the number one A... Continue Bio >>
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The Motels Wang Chung Juice Newton Taylor Dayne Bonnie Tyler Scandal Kim Wilde Wilson Phillips Olivia Newton-John Steve Perry
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COMMERCIAL DESCRIPTION Brewed since the beginning of last century, when the near-complete staff of the brewery went to see the circus of Buffalo Bill, leaving the boiling brewing kettle to the care of the youngest employee. This boy let the beer roast and caramelise on the bottom of the kettle, but after trying, everybody actually liked the "spoiled" beer, which became the brewerys staple diet. This Belgian version is a lot weaker than the stout exported to the USA.
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European Journal of Plant Pathology
The European Journal of Plant Pathology is an international journal that publishes original research articles dealing with fundamental and applied aspects of plant pathology. Thus, in addition to bacteriological, mycological, and virological topics, entomological, nematological and plant protection studies in general are also included. 5-Year Impact Factor: 2.054
138 Volumes 858 Issues 6,106 Articles available from 1895 - 2014
Browse Volumes & IssuesLatest Articles
Penetration and reproduction of root-knot nematodes on cucurbit species
Manuel López-Gómez, Soledad Verdejo-Lucas (April 2014)
Biological control of avocado white root rot with combined applications of Trichoderma spp. and rhizobacteria
D. Ruano-Rosa, F. M. Cazorla, N. Bonilla… (April 2014)
Original Research
Effect of Avocado sunblotch viroid (ASBVd) on avocado yield in Michoacan, Mexico
J. R. Saucedo-Carabez, D. Téliz-Ortiz… (April 2014)
- Journal Title
- European Journal of Plant Pathology
- Coverage
- Volume 1 / 1895 - Volume 138 / 2014
- Print ISSN
- 0929-1873
- Online ISSN
- 1573-8469
- Publisher
- Springer Netherlands
- Additional Links
- Topics
- Industry Sectors
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