Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire

Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells that required HLA-B*57:01 molecules for their function. However, the mechanism by which abacavir induces this pathologic T cell response remains unclear. Here we show that abacavir can bind within the F-pocket of the peptide-binding groove of HLA-B*57:01 thereby altering its specificity. This supports a novel explanation for HLA-linked idiosyncratic adverse drug reactions; namely that drugs can alter the repertoire of self-peptides presented to T cells thus causing the equivalent of an alloreactive T cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir, and that were recognized by T cells of hypersensitive patients. The assays we have established can be applied to test additional compounds with suspected HLA linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA linked hypersensitivities as well as guide the development of safer drugs

Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan

CD4 positive T helper cells control many aspects of specific immunity. These cells are specific for peptides derived from protein antigens and presented by molecules of the extremely polymorphic major histocompatibility complex (MHC) class II system. The identification of peptides that bind to MHC class II molecules is therefore of pivotal importance for rational discovery of immune epitopes. HLA-DR is a prominent example of a human MHC class II. Here, we present a method, NetMHCIIpan, that allows for pan-specific predictions of peptide binding to any HLA-DR molecule of known sequence. The method is derived from a large compilation of quantitative HLA-DR binding events covering 14 of the more than 500 known HLA-DR alleles. Taking both peptide and HLA sequence information into account, the method can generalize and predict peptide binding also for HLA-DR molecules where experimental data is absent. Validation of the method includes identification of endogenously derived HLA class II ligands, cross-validation, leave-one-molecule-out, and binding motif identification for hitherto uncharacterized HLA-DR molecules. The validation shows that the method can successfully predict binding for HLA-DR molecules-even in the absence of specific data for the particular molecule in question. Moreover, when compared to TEPITOPE, currently the only other publicly available prediction method aiming at providing broad HLA-DR allelic coverage, NetMHCIIpan performs equivalently for alleles included in the training of TEPITOPE while outperforming TEPITOPE on novel alleles. We propose that the method can be used to identify those hitherto uncharacterized alleles, which should be addressed experimentally in future updates of the method to cover the polymorphism of HLA-DR most efficiently. We thus conclude that the presented method meets the challenge of keeping up with the MHC polymorphism discovery rate and that it can be used to sample the MHC "space," enabling a highly efficient iterative process for improving MHC class II binding predictions

Microscopic dynamics in liquid metals: the experimental point of view

The experimental results relevant for the understanding of the microscopic dynamics in liquid metals are reviewed, with special regards to the ones achieved in the last two decades. Inelastic Neutron Scattering played a major role since the development of neutron facilities in the sixties. The last ten years, however, saw the development of third generation radiation sources, which opened the possibility of performing Inelastic Scattering with X rays, thus disclosing previously unaccessible energy-momentum regions. The purely coherent response of X rays, moreover, combined with the mixed coherent/incoherent response typical of neutron scattering, provides enormous potentialities to disentangle aspects related to the collectivity of motion from the single particle dynamics. If the last twenty years saw major experimental developments, on the theoretical side fresh ideas came up to the side of the most traditional and established theories. Beside the raw experimental results, therefore, we review models and theoretical approaches for the description of microscopic dynamics over different length-scales, from the hydrodynamic region down to the single particle regime, walking the perilous and sometimes uncharted path of the generalized hydrodynamics extension. Approaches peculiar of conductive systems, based on the ionic plasma theory, are also considered, as well as kinetic and mode coupling theory applied to hard sphere systems, which turn out to mimic with remarkable detail the atomic dynamics of liquid metals. Finally, cutting edges issues and open problems, such as the ultimate origin of the anomalous acoustic dispersion or the relevance of transport properties of a conductive systems in ruling the ionic dynamic structure factor are discussed.Comment: 53 pages, 41 figures, to appear in "The Review of Modern Physics". Tentatively scheduled for July issu

Driven Assembly of Lignin into Microcapsules for Storage and Delivery of Hydrophobic Molecules

Oil-filled microcapsules of kraft lignin were synthe- sized by first creating an oil in water emulsion followed by a high- intensity, ultrasound-assisted cross-linking of lignin at the water/oil interface. The rationale behind our approach is based on promoting documented lignin hydrophobic interactions within the oil phase, followed by locking the resulting spherical microsystems by covalent cross-linking using a high intensity ultrasound treatment. As further evidence in support of our rationale, confocal and optical microscopies demonstrated the uniformly spherical morphology of the created lignin microparticles. The detailed elucidation of the cross-linking processes was carried out using gel permeation chromatography (GPC) and quantitative 31P NMR analyses. The ability of lignin microcapsules to incorporate and release Coumarin-6 was evaluated in detail. In vitro studies and confocal laser scanning microscopy analysis were carried out to assess the internalization of capsules into Chinese hamster ovary (CHO) cells. This part of our work demonstrated that the lignin microcapsules are not cytotoxic and readily incorporated in the CHO cells

Design and utilization of epitope-based databases and predictive tools

In the last decade, significant progress has been made in expanding the scope and depth of publicly available immunological databases and online analysis resources, which have become an integral part of the repertoire of tools available to the scientific community for basic and applied research. Herein, we present a general overview of different resources and databases currently available. Because of our association with the Immune Epitope Database and Analysis Resource, this resource is reviewed in more detail. Our review includes aspects such as the development of formal ontologies and the type and breadth of analytical tools available to predict epitopes and analyze immune epitope data. A common feature of immunological databases is the requirement to host large amounts of data extracted from disparate sources. Accordingly, we discuss and review processes to curate the immunological literature, as well as examples of how the curated data can be used to generate a meta-analysis of the epitope knowledge currently available for diseases of worldwide concern, such as influenza and malaria. Finally, we review the impact of immunological databases, by analyzing their usage and citations, and by categorizing the type of citations. Taken together, the results highlight the growing impact and utility of immunological databases for the scientific community

PTEN status is a crucial determinant of the functional outcome of combined MEK and mTOR inhibition in cancer

Combined MAPK/PI3K pathway inhibition represents an attractive, albeit toxic, therapeutic strategy in oncology. Since PTEN lies at the intersection of these two pathways, we investigated whether PTEN status determines the functional response to combined pathway inhibition. PTEN (gene, mRNA, and protein) status was extensively characterized in a panel of cancer cell lines and combined MEK/mTOR inhibition displayed highly synergistic pharmacologic interactions almost exclusively in PTEN-loss models. Genetic manipulation of PTEN status confirmed a mechanistic role for PTEN in determining the functional outcome of combined pathway blockade. Proteomic analysis showed greater phosphoproteomic profile modification(s) in response to combined MEK/mTOR inhibition in PTEN- loss contexts and identified JAK1/STAT3 activation as a potential mediator of synergistic interactions. Overall, our results show that PTEN-loss is a crucial determinant of synergistic interactions between MAPK and PI3K pathway inhibitors, potentially exploitable for the selection of cancer patients at the highest chance of benefit from combined therapeutic strategies

Generation of dendritic cell-based vaccines for cancer therapy

Dendritic cells play a major role in the generation of immunity against tumour cells. They can be grown under various culture conditions, which influence the phenotypical and functional properties of dendritic cells and thereby the consecutive immune response mainly executed by T cells. Here we discuss various conditions, which are important during generation and administration of dendritic cells to elicit a tumouricidal T cell-based immune response

Proteome Sampling by the HLA Class I Antigen Processing Pathway

The peptide repertoire that is presented by the set of HLA class I molecules of an individual is formed by the different players of the antigen processing pathway and the stringent binding environment of the HLA class I molecules. Peptide elution studies have shown that only a subset of the human proteome is sampled by the antigen processing machinery and represented on the cell surface. In our study, we quantified the role of each factor relevant in shaping the HLA class I peptide repertoire by combining peptide elution data, in silico predictions of antigen processing and presentation, and data on gene expression and protein abundance. Our results indicate that gene expression level, protein abundance, and rate of potential binding peptides per protein have a clear impact on sampling probability. Furthermore, once a protein is available for the antigen processing machinery in sufficient amounts, C-terminal processing efficiency and binding affinity to the HLA class I molecule determine the identity of the presented peptides. Having studied the impact of each of these factors separately, we subsequently combined all factors in a logistic regression model in order to quantify their relative impact. This model demonstrated the superiority of protein abundance over gene expression level in predicting sampling probability. Being able to discriminate between sampled and non-sampled proteins to a significant degree, our approach can potentially be used to predict the sampling probability of self proteins and of pathogen-derived proteins, which is of importance for the identification of autoimmune antigens and vaccination targets