ALBERT

Beschreibung: Key Points High, but not low to moderate, HLA antibody levels are associated with platelet refractoriness.

Beschreibung: Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients. Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed. Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p

Beschreibung: Abstract 220 Introduction. The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages. Aims: (1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles. Methods. The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined. Results. Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 〉 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency). Conclusions. MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential. Disclosures: Pratt: CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.

Beschreibung: Abstract 1128 Evidence first emerged in the 1930s that omega-3 (n-3) and omega-6 (n-6) fatty acids (FA) were important for normal health and growth. A 1960s study of Greenland Inuits provided the first evidence that dietary intake of n-3 FA correlated with a reduction in cardiovascular disease. Omega-3 FA (eicosapentaenoic acid [EPA] and docosahexanoic acid [DHA]), along with n-6 FA (chiefly linoleic acid) are fats essential for health, with a minimum recommended intake of 0.2% and 1% of daily calories, respectively; and a healthy ratio of n-6 to n-3 FAs of 1:1 to 1:4. Increased consumption of n-3 FA has recently been shown to correlate with a reduced death rate from coronary heart disease in both healthy individuals and those with pre-existing cardiovascular disease. Cardiovascular diseases, including myocardial infarction and stroke, are precipitated by pathological platelet thrombus formation and are the leading causes of mortality and morbidity in developed countries. In both thrombosis and hemostasis, platelets attach to areas of vessel injury by binding to von Willebrand Factor (VWF) at the injury site. The platelet receptor for VWF is the glycoprotein (GP) Ib-IX-V complex, specifically GPIbα within the complex. This interaction also mediates fluid-phase platelet aggregation in regions of elevated hydrodynamic shear stress, a process called shear-induced platelet aggregation (SIPA). Glycoprotein Ib-IX-V complex functions depend on its localization to lipid raft domains within the plasma membrane, localization mediated by palmitoylation of Cys residues and involving sequences in the subunit transmembrane and cytoplasmic domains. We reasoned that by interfering with palmitoylation or disrupting lipid rafts, or through both mechanisms, n-3 FAs would decrease platelet functions. We therefore studied the effect of n-3 FAs on platelet functions in 12 healthy adult volunteers who ingested 2400 mg of EPA + DHA daily for 2 months. Blood was drawn from the subjects before they began ingesting n-3 FAs (at weeks -4,-3, and -1), then again during the dosing period (weeks 5, 6, and 8). We assessed complete blood counts, and platelet aggregation in response to ADP (2 μM and 20 μM), arachadonic acid (ACA) (250 μg/ml and 500 μg/ml), and shear stress (at shear rates of 2000, 5000 and 10000 s−1). We also assessed GPIb-IX-V lipid raft localization. In the majority of subjects, platelet aggregation after n-3 FA treatment was reduced with low-concentrations of ADP but not with ACA. The most striking effect was on SIPA, which was reduced by 90% (±2% SEM) at 2000 s−1 and 30% (±7% SEM) at 5000 s−1. No post-treatment difference in SIPA was observed at 10000 s−1. After intake of n-3 FAs, the lipid raft content of GPIb-IX-V decreased by 50% while two raft markers, GM1 ganglioside and flotillin, remained unchanged. These findings indicate that n-3 FAs likely suppress several platelet functions, with one of the most striking being decreased adhesive activity, which correlates with decreased raft content of GPIb-IX-V. Thus, the anti-thrombotic effect of n-3 FAs results from a combination of diminished response to some soluble platelet agonists and reduced adhesive capacity. Disclosures: No relevant conflicts of interest to declare.