Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

‘How can I be post-Soviet if I was never Soviet?’ Rethinking categories of time and social change – a perspective from Kulob, southern Tajikistan

Based on anthropological fieldwork conducted in the Kulob region of southern Tajikistan, this paper examines the extent to which the existing periodization ‘Soviet/post-Soviet’ is still valid to frame scholarly works concerning Central Asia. It does so through an analysis of ‘alternative temporalities’ conveyed by Kulob residents to the author. These alternative temporalities are fashioned in especially clear ways in a relationship to the physical transformations occurring to two types of housing, namely flats in building blocks and detached houses. Without arguing that the categories ‘Soviet’ and ‘post-Soviet’ have become futile, the author advocates that the uncritically use of Soviet/post-Soviet has the unwanted effect of shaping the Central Asian region as a temporalized and specialized ‘other’

Stability and sensory assessment of emulsions containing propolis extract and/or tocopheryl acetate

The prevention of skin aging has been one of the main aims of cosmetic products. Propolis and tocopheryl acetate can be promising substances because of their antioxidant properties. In this study, propolis extract was obtained and associated with tocopheryl acetate in a cream formulation, which then underwent stability and sensory assessment. The formulation containing propolis extract and tocopheryl acetate proved to be stable in the preliminary stability study, demonstrating gradual darkening and slight pH decrease when subjected to 60ºC for 28 days, but showing stability on rheological study. In the sensory analysis, the formulation containing these two components was preferred by the product testers over the base cream and creams containing propolis extract or tocopheryl acetate alone. In conclusion, given the stability of the formulation and the preference of the product testers for this formulation, this association proved promising for use in cosmetic formulations.A prevenção do envelhecimento cutâneo tem sido um dos principais focos dos produtos dermocosméticos. A própolis contém substâncias com atividade antioxidante, bem como o acetato de tocoferila é conhecido por apresentar esta atividade. Porém, a própolis apresenta odor muito característico e intenso, que pode interferir no sensorial do produto. Assim, no presente trabalho, obteve-se o extrato de própolis, que foi associado ao acetato de tocoferila em uma formulação de uso tópico, que foi avaliada quanto à estabilidade e às características sensoriais. Conduziu-se um estudo de estabilidade, no qual as formulações contendo ambos os compostos apresentaram escurecimento gradual e ligeira queda no pH após 28 dias sob 60 °C, tendo sido estável no estudo reológico. Na análise sensorial, realizada com 28 provadores, a formulação contendo extrato de própolis em associação com acetato de tocoferila foi a preferida, quando comparada com o creme base e o creme contendo somente extrato de própolis ou acetato de tocoferila. Em conclusão, devido à preferência dos provadores e ao estudo de estabilidade, a associação de extrato de própolis e de acetato de tocoferila mostrou ser promissora para uso em produtos dermocosméticos

Postcolonial manifestations of African spatiality in Europe : the invisible 'public' spaces of Ghent

The focus of this chapter is on everyday spaces of African migration in the mid-sized city of Ghent. One manifestation of African spatiality is discussed in-depth in relation to its (in)visibility and publicity: an African shop located in an ordinary terraced house. With no less than 12 activities taking place in the building, the shop is rather a “public” place than solely a space of commercial transactions, although this is not signaled in very visible ways. By analyzing the modest stylistic appropriations of the façade and the significant re-arrangements of the buildings’ interior spaces that challenge more conventional usages of spaces in Ghent’s ordinary houses, this chapter puts this African shop to the fore as emblematic of how the process of materialization of transnational lifestyles and connections is always a balancing act between the visibility necessary for functioning as a (semi-)pubic place and the invisibility required to circumvent hegemonic regulatory regimes

Learning Legged Locomotion

Legged locomotion of biological systems can be viewed as a self-organizing process of highly complex system-environment interactions. Walking behavior is, for example, generated from the interactions between many mechanical components (e.g. physical interactions between feet and ground, skeletons an

Nogo-A Expression in the Brain of Mice with Cerebral Malaria

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM

Characterization of Structural Features Controlling the Receptiveness of Empty Class II MHC Molecules

MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying “non-receptiveness.” Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the α-helix of the β1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues αQ9 and βN82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting from the structural knowledge provided by this study