Programmed remodeling of cell surface glycans by the sequential action of specific glycosyltransferases, can control biological processes by generating or masking ligands for endogenous lectins. Galectins, a family of animal lectins with affinity for β-galactosides, can form multivalent complexes with cell surface glycoconjugates and deliver a variety of intracellular signals to modulate cell activation, differentiation, and survival. Recent efforts involving genetic or biochemical manipulation of O-and N-glycosylation pathways, as well as blockade of the synthesis of endogenous galectins, have illuminated essential roles for galectin-glycoprotein lattices in the control of biological processes including receptor turnover and endocytosis, host-pathogen interactions and immune cell activation and homeostasis.

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Virus-specific CD4+ T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4+ as well as CD8+ T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4+ T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4+ T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4+ T cell responses was selective, since vaccine-elicited Ab and CD8+ T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4+ T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8؉ T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8 ؉ T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.Replication-defective recombinant adenovirus serotype 5 (rAd5) vector-based vaccines elicit potent and protective immune responses in a variety of animal models (4,19,22,23). Clinical trials of rAd5 vaccines for human immunodeficiency virus type 1 (HIV-1) and other pathogens are therefore currently in progress (14). A major limitation of this approach, however, is that the majority of the human population has preexisting anti-Ad5 immunity that may substantially reduce the immunogenicity and clinical utility of rAd5 vaccines. In fact, anti-Ad5 immunity has already been demonstrated to suppress the immunogenicity of rAd5 vaccines in studies in mice (3, 31), rhesus monkeys (4), and humans in early phase I clinical trials.It is generally accepted that potent Ad5-specific neutralizing antibodies (NAbs) contribute substantially to the suppressive effects of anti-Ad5 immunity (7,8,12,25,27,32). Far less is known about the biological importance of Ad5-specific T-lymphocyte responses. Ad5-specific CD4 ϩ and CD8 ϩ T lymphocytes have been found in both humans (9,17,18,20) and mice (11,13,21,29,30). However, the importance of Ad5-specific T lymphocytes in suppressing the immunogenicity of rAd5 vaccines has not been fully characterized.In this study, we investigate the relative contributions of Ad5-specific humoral and cellular immune responses in suppressing the immunogenicity of a rAd5-Env vaccine in mice. By adoptive-transfer studies, we demonstrate that NAbs and CD8 ϩ T lymphocytes both contribute to immunity to Ad5. These data suggest that novel Ad vector-based vaccines should overcome both humoral and cellular immune responses to circumvent preexisting anti-Ad5 immunity. MATERIALS AND METHODSMice and immunizations. BALB/c mice, 6 to 8 weeks old, were purchased from Charles River Laboratories (Wilmington, Mass.). ...

In Arabidopsis, a complex of Polycomb-group (PcG) proteins functions in the female gametophyte to control the initiation of seed development. Mutations in the PcG genes, including MEDEA (MEA) and FERTILIZATION-INDEPENDENT SEED 2 (FIS2), produce autonomous seeds where endosperm proliferation occurs in the absence of fertilization. By using a yeast two-hybrid screen, we identified MEA and a related protein, SWINGER (SWN), as SET-domain partners of FIS2. Localization data indicated that all three proteins are present in the female gametophyte. Although single-mutant swn plants did not show any defects, swn mutations enhanced the mea mutant phenotype in producing autonomous seeds. Thus, MEA and SWN perform partially redundant functions in controlling the initiation of endosperm development before fertilization in Arabidopsis.chromatin ͉ endosperm ͉ female gametophyte ͉ fertilization ͉ gene silencing I n angiosperms, seed development is initiated by a doublefertilization event within the female gametophyte (FG). The pollen tube delivers two sperm cells that fuse with the haploid egg cell and the typically homodiploid central cell to initiate the development of two distinct organs, an embryo and an endosperm, respectively (1). The diploid embryo forms the next generation, whereas the typically triploid endosperm is a shortlived nutritive tissue that supports embryogenesis or seedling development. In most angiosperms, including Arabidopsis, the endosperm undergoes a nuclear-type development that includes an initial period of coenocytic development in which the primary endosperm nucleus undergoes mitosis without cytokinesis followed by cellularization. In Arabidopsis, the cellularized endosperm is gradually absorbed by the developing embryo, and only an aleurone layer remains at seed maturity (2, 3).Recent evidence indicates that the FG controls the initiation of seed development in Arabidopsis. Mutations in genes encoding the Polycomb-group (PcG) proteins FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), MEDEA (MEA), FERTILIZATION-INDEPENDENT SEED 2 (FIS2), and the Arabidopsis homologue of MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) cause the development of autonomous seeds in the absence of fertilization due mainly to proliferation of the diploid central cell nucleus. Molecular and genetic evidence indicates that these proteins are expressed in the FG and function to repress downstream genes required for normal endosperm proliferation before fertilization (4-11). The FIE, MEA, FIS2, and MSI1 genes encode proteins that are highly similar to components of the Polycomb repressive complex 2͞3 (PRC2͞3) that include the Drosophila Esc, E(z), Su(z)12, and p55 and the mammalian EED, EZH2, SUZ12, and RbAp48͞46, respectively (12). PRC2͞3 and the larger PRC1 complex function to silence and maintain epigenetic silencing of target genes involved in regulation of early development, cell growth, and proliferation. At least part of the gene-silencing function of PRC2͞3 is likely mediated through its intrinsic histone H3 methylation activity (13, 1...

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.Priming with plasmid DNA vaccines and boosting with recombinant viral vectors such as replication-defective adenoviruses and poxviruses have been shown to generate potent virus-specific cytotoxic T-lymphocyte (CTL) responses (2,9,11,17,19). Since virus-specific CTL responses are critical for the control of human immunodeficiency virus type 1 (HIV-1) (6, 10, 13, 15), these "prime-boost" strategies are being assessed as candidate AIDS vaccines (12). Recent studies have demonstrated that DNA prime-replication-defective adenovirus serotype 5 (rAd5) boost regimens (17) as well as DNA primerecombinant-modified vaccinia virus Ankara (rMVA) boost regimens (2) afford significant control of pathogenic simianhuman immunodeficiency virus (SHIV) challenges in rhesus monkeys. Similarly, DNA prime-rAd5 boost regimens have protected nonhuman primates against lethal challenge with Ebola virus (19).The clinical utility of such prime-boost regimens, however, will likely be limited by preexisting immunity to the viral vector. Antivector immunity may result in the rapid elimination of the vaccine vector and thus could substantially reduce its immunogenicity. This problem is expected to be a major limitation for rAd5 vectors, since a large fraction of the human population has preexisting anti-Ad5 immunity as a result of natural exposure to Ad5. Strategies to improve these vaccine regimens are therefore needed.One potential strategy for improving the immunogenicity of DNA prime-viral vector boost vaccine regimens is to improve the efficiency of the DNA prime by the coadministration of adjuvants. Our laboratory and others have previously demonstrated that plasmid cytokines can augment DNA vaccineelicited humoral and cellular immune responses in both mice and rhesus monkeys (4-6, 16, 21, 23). Here we investigate the utility of chemoattractant plasmid cytokine...

Delirium is not only one of the most common complications that older patients develop after admission to hospital but it is also one of the most serious. Although stroke is a known predisposing factor for delirium, few studies have investigated this association and results from existing studies give conflicting results with prevalence estimates ranging from 13 to 48%. The aetiology of delirium post-stroke is poorly understood. There is no consensus on the best screening tool to use to detect delirium in the post-stroke setting. Specific stroke types may be more likely to precipitate delirium than others, for example, delirium is more frequent after intracerebral haemorrhage and total anterior circulation infarction (TACI). In addition, case reports have suggested that delirium may be associated with specific lesions, for example, in the thalamus and caudate nucleus. There is a lack of intervention data in both the prevention and treatment of delirium post-stroke. However, it is known that the development of delirium post-stroke has grave prognostic implications. It is associated with longer stay in hospital, increased mortality and increased risk of institutionalisation post discharge. In this article, we review the literature to date on delirium in the acute stroke setting.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope ± cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1α plus IL-18, IL-1α plus IL-12, and IL-1α plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60–71% peptide-specific 51Cr release). Peak H-2Dd-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8+ PBMC. Nasal immunization with HIV immunogen and IL-1α, IL-12, and GM-CSF also induced Ag-specific IFN-γ-secreting cells in the draining cervical lymph node and the lung. The use of IL-1α, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1α, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.