Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)–associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor’s MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although .12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)–recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain–deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/ present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII

The Development of FVIII Inhibitor in Hispanic American Patients with Hemophilia A Critically Impacts Coagulation Potential

Background: Hemophilia A (HA) is caused by deficiencies in plasma-FVIII and heterogeneous factor-VIII-gene mutations that impair intrinsic coagulation amplification. In severe hemophilia A patients (HAPs), FVIII infusions are begun at toddlerhood to prevent hemarthrosis induced crippling. However, approximately 30% of these patients develop FVIII inhibitors. Gain-of-function mutations in the common pathway of coagulation increases coagulation potential and decreases bleeding and FVIII-utilization in HAPs which should decrease FVIII-inhibitor-risk. We identified loss-of-function mutations in this pathway which decrease coagulation-potential as they increase FVIII-inhibitor risk in HAPs. Methods: We screened Mexican-American-pedigrees of the South-Texas-Family-Study (STFS) for protein-altering-variants. Subjects were genotyped using Illumina-exome-24-chip. Protein-altering-variants were analyzed for associations with FII:C, PT, and aPTT. Linear-mixed-model-analyses was performed to estimate trait-heritability and examine single-nucleotide-variations (SNVs) for gene association. Significant associations’ p-values fell below Bonferroni-adjusted significance level. Results: Heritability-estimates for FII:C, aPTT, and PT were highly-significant with p-values of 0.49, 0.49, and 0.54 (for all, pT in the FII-gene (F2)—which encodes 543R\u3eL and has a large effect-size on each trait (for all, pT have lower FII:C levels but correspondingly prolonged aPTT and PT times. Conclusion: We hypothesize that FII-543R\u3eL (Prothrombin-RGV) likely contributes to the high-incidence of FVIII-inhibitor-development in HA-patients of Mexican-ancestry, resulting in higher risk of developing anti-tFVIII-antibodies than patients without the variant. Patients with the RGV variant are likely to bleed more which can require surgery, further increasing the development of FVIII inhibitor development

One health, une seule santé

One Health, « Une seule santé », est une stratégie mondiale visant à développer les collaborations interdisciplinaires pour la santé humaine, animale et environnementale. Elle promeut une approche intégrée, systémique et unifiée de la santé aux échelles locale, nationale et mondiale, afin de mieux affronter les maladies émergentes à risque pandémique, mais aussi s'adapter aux impacts environnementaux présents et futurs. Bien que ce mouvement s’étende, la littérature en français reste rare. Traduit de l’anglais, coordonné par d’éminents épidémiologistes et s'appuyant sur un large panel d' approches scientifiques rarement réunies autour de la santé, cet ouvrage retrace les origines du concept et présente un contenu pratique sur les outils méthodologiques, la collecte de données, les techniques de surveillance et les plans d’étude. Il combine recherche et pratique en un seul volume et constitue un ouvrage de référence unique pour la santé mondiale

Quantitative HLA-class-II/factor VIII (FVIII) peptidomic variation in dendritic cells correlates with the immunogenic potential of therapeutic FVIII proteins in hemophilia A

Background Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. Objectives Use dendritic cell (DC)-protein processing/presentation assays with mass-spectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, and DR-bound FVIII-derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. Patients/Methods Monocyte-derived DCs from normal donors and/or PWHA were cultured with either: Mix-rFVIII, a VWF-free equimolar mixture of a full-length (FL)-rFVIII [Advate® (Takeda)] and four distinct B-domain-deleted (BDD)-rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo-Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. Results We showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla − pdVWF, and Mix-rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. Conclusions Our results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field