Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the ß5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

Acceptance of health technology assessment submissions with incremental cost-effectiveness ratios above the cost-effectiveness threshold.

OBJECTIVES: In health technology assessment (HTA) agencies where cost-effectiveness plays a role in decision-making, an incremental cost-effectiveness ratio (ICER) threshold is often used to inform reimbursement decisions. The acceptance of submissions with ICERs higher than the threshold was assessed across different agencies and across indications, in order to inform future reimbursement submissions. METHODS: All HTA appraisals from May 2000 to May 2014 from National Institute for Health and Care Excellence (NICE), Scottish Medicines Consortium (SMC), Pharmaceutical Benefits Advisory Committee (PBAC), and Canadian Agency for Drugs and Technologies in Health (CADTH) were assessed. Multiple technology appraisals, resubmissions, vaccination programs, and requests for advice were excluded. Submissions not reporting an ICER, or for which an ICER could not be determined were also excluded. The remaining appraisals were reviewed, and the submitted ICER, recommendation, and reasoning behind the recommendation were extracted. RESULTS: NICE recommended the highest proportion of submissions with ICERs higher than the threshold (34% accepted without restrictions; 20% with restrictions), followed by PBAC (16% accepted without restrictions; 4% with restrictions), SMC (11% accepted without restrictions; 14% accepted with restrictions), and CADTH (0% accepted without restrictions; 26% with restrictions). Overall, the majority of higher-than-threshold ICER submissions were classified into the "malignant disease and immunosuppression" therapeutic category; however, there was no notable variation in acceptance rates by disease area. Reasons for accepting submissions reporting ICERs above the threshold included high clinical benefit over the standard of care, and addressing an unmet therapeutic need. CONCLUSION: Acceptance of submissions with higher-than-threshold ICERs varied by HTA agency and was not significantly influenced by disease category. Such submissions must be accompanied by robust, concrete, and transparent evidence in order to achieve patient access.

Therapeutic potential of larval excretory/secretory proteins of the pig whipworm Trichuris suis in allergic disease.

BACKGROUND: Gastrointestinal nematodes are currently being evaluated as a novel therapeutic in the treatment of chronic human inflammatory disorders, due to their unique ability to induce immunoregulatory pathways in their hosts. In particular, administration of ova from the pig whipworm Trichuris suis (T. suis; TSO) has been proposed for the treatment of allergic, inflammatory and autoimmune disorders. Despite these advances, the biological pathways through which TSO therapy modulates the host immune system in the context of human disease remain undefined. METHODS: We characterized the dominant proteins present in the excretory/secretory (E/S) products of first-stage (L1) T. suis larvae (Ts E/S) using LC-MS/MS analysis and examined the immunosuppressive properties of whole larval Ts E/S in vitro and in a murine model of allergic airway disease. RESULTS: Administration of larval Ts E/S proteins in vivo during the allergen sensitization phase was sufficient to suppress airway hyperreactivity, bronchiolar inflammatory infiltrate and allergen-specific IgE production. Three proteins in larval Ts E/S were unambiguously identified. The immunomodulatory function of larval Ts E/S was found to be partially dependent on the immunoregulatory cytokine IL-10. CONCLUSIONS: Taken together, these data demonstrate that the released proteins of larval T. suis have significant immunomodulatory capacities and efficiently dampen allergic airway hyperreactivity. Thus, the therapeutic potential of defined larval E/S proteins should be exploited for the treatment of human allergic disorders.

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues.

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.

Characterization of intermolecular beta-sheet peptides by mass spectrometry and hydrogen isotope exchange.

The self-assembly of beta-sheet peptide domains resulting in the formation of fibrillar aggregates (amyloids) is a feature of various neurodegenerative disorders. In order to evaluate mass spectrometric methods for the characterization of intermolecular beta-sheet structures the hydrogen/deuterium exchange behaviour of model peptides DPKGDPKG-(VT)(n)-GKGDPKPD-amide (n = 3,4,5,6,7,8), (VT)(n)-peptides, composed of a central beta-sheet-forming domain and N- and C-terminal nonstructured octapeptide sequences, was measured by electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kinetic analysis of the hydrogen/deuterium exchange (HX) shows that intermolecular beta-sheet structures contain slowly exchanging protons (k

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I).

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith applies were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients' sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Furthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.