Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2,3 and 6 in a mouse model for peanut allergy.

BACKGROUND: The relative contribution and the relation between individual peanut allergens in peanut allergic responses is still matter of debate. We determined the individual contribution of peanut proteins to B, T cell and allergic effector responses in a mouse model for peanut allergy. METHODS: Mice were immunized and challenged by oral gavage with peanut protein extract or isolated allergens Ara h 1, 2, 3 and 6 followed by assessment of food allergic manifestations. In addition, T cell responses to the individual proteins were measured by an in vitro dendritic cell-T cell assay. RESULTS: Sensitization with the individual peanut proteins elicited IgE responses with specificity to the allergen used as expected. However, cross reactivity among Ara h 1, 2, 3 and 6 was observed. T cell re-stimulations with peanut extract and individual peanut proteins also showed cross reactivity between Ara h 1, 2, 3 and 6. Despite the cross reactivity at the IgE level, only Ara h 2 and 6 were able to elicit mast cell degranulation after an oral challenge. However, after systemic challenge, Ara h 1, 2 and 6 and to lesser extent Ara h 3 were able to elicit anaphylactic responses. CONCLUSIONS: Ara h 1, 2, 3 and 6 sensitize via the intra-gastric route, but differ in their capacity to cause allergic effector responses. Interestingly, extensive cross reactivity at T cell and antibody level is observed among Ara h 1, 2, 3 and 6, which may have important implications for the diagnosis and therapy of peanut allergy. Awareness about the relative contribution of individual peanut allergens and cross reactivity between these allergens is of importance for current research in diagnostics and therapeutics for and the mechanism of peanut allergy.

Optimisation of lupus anticoagulant tests: should test samples always be mixed with normal plasma?

Coagulation factor deficiencies are thought to interfere with the detection of the phospholipid-dependent coagulation inhibitor known as lupus anticoagulant (LA). Treatment with vitamin K antagonists (VKA) in particular, is thought to preclude accurate LA assessment. For this reason, the procedure to detect LA includes a mixing test, in which coagulation factor deficiencies are corrected by mixing samples with an equal volume of normal plasma. Despite these mixing tests, interpretation of LA test results is considered difficult in patients receiving high intensity VKA treatment. As a result, VKA treatment is often temporarily discontinued to allow LA assessment. However, whether coagulation factor deficiencies influence LA test results is unclear. We found that neither deficiency of a single coagulation factor, nor a functional coagulation factor deficiency due to high intensity VKA treatment, resulted in false positive dRVVT- or APTT-based (silica clotting time; SCT) LA test results. LA was readily detected in unmixed samples from VKA-treated LA-positive patients with both dRVVT and SCT reagents. VKA treatment caused an underestimation of the strength of the LA with SCT reagents, but did not lead to misclassification of LA status. Although mixing with normal plasma during both screen and confirm tests allowed more accurate assessment of the strength of the LA with SCT reagents in samples with an international normalised >2.5, the mixing procedure itself lead to misclassification of LA in weakly positive samples from patients not treated with VKA. Based on these findings, we conclude that mixing studies are not necessary during LA-assessment.

Apolipoprotein E receptor 2 is involved in the thrombotic complications in a murine model of the antiphospholipid syndrome.

Antiphospholipid (aPL)/anti-ß(2) glycoprotein I (anti-ß(2)GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2') on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-ß(2)GPI monoclonal antibody (E7) and of a constructed dimeric ß(2)GPI I (dimer), which in vitro mimics ß(2)GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (-/-) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (-/-) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.

Pathogenic anti-beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after a conformational change.

Recently, we published the existence of 2 populations of anti-beta2-glycoprotein I (beta2-GPI) IgG antibodies. Type A antibodies recognize epitope G40-R43 in domain I of beta2-GPI and are strongly associated with thrombosis. Type B antibodies recognize other parts of beta2-GPI and are not associated with thrombosis. In this study we demonstrate that type A antibodies only recognize plasma-purified beta2-GPI when coated onto a negatively charged surface and not when coated onto a neutrally charged surface. The affinity of type B antibodies toward plasma-purified beta2-GPI was independent of the charge of the surface to which beta2-GPI was coated. Type A antibodies did not recognize plasma-purified beta2-GPI in solution, whereas they did recognize recombinant beta2-GPI both in solution and coated onto a neutrally charged plate. When the carbohydrate chains were removed from plasma-purified beta2-GPI, we found that type A antibodies did recognize the protein in solution. This supports the hypothesis that the difference in recognition of plasma-purified and recombinant beta2-GPI is caused by the difference in glycosylation and that epitope G40-R43 of plasma-purified beta2-GPI is covered by a carbohydrate chain. Type A anti-beta2-GPI antibodies can only recognize this epitope when this carbohydrate chain is displaced as a result of a conformational change. This finding has major implications both for the detection of pathogenic anti-beta2-GPI antibodies and the comprehension of the pathophysiology of the antiphospholipid syndrome.

The binding site in {beta}2-glycoprotein I for ApoER2' on platelets is located in domain V.

The antiphospholipid syndrome is caused by autoantibodies directed against beta(2)-glycoprotein I (beta(2)GPI). Dimerization of beta(2)GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2' (apoER2'), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric beta(2)GPI. To identify which domain of dimeric beta(2)GPI interacts with apoER2', we have constructed domain deletion mutants of dimeric beta(2)GPI, lacking domain I (DeltaI), II (DeltaII), or V (DeltaV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. DeltaI and DeltaII prolonged the clotting time, as did full-length dimeric beta(2)GPI; DeltaV had no effect on the clotting time. Second, DeltaI and DeltaII bound to anionic PL, comparable with full-length dimeric beta(2)GPI. DeltaV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric beta(2)GPI, DeltaI, or DeltaII (mean increase 150%) were added to whole blood. No increase was found with plasma beta(2)GPI, DeltaV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric beta(2)GPI, DeltaI, DeltaII, and the W316S mutant can interact with apoER2' on platelets. DeltaV did not associate with apoER2'. We conclude that domain V is involved in both binding beta(2)GPI to anionic PL and in interaction with apoER2' and subsequent activation of platelets. The binding site in beta(2)GPI for interaction with apoER2' does not overlap with the hydrophobic insertion loop in domain V.

The lipopolysaccharide biosynthesis locus of Campylobacter jejuni 81116.

Most Campylobacter jejuni strains express lipo-oligosaccharides. Some strains also express lipopolysaccharides (LPS), with O-antigen-like carbohydrate repeats. C. jejuni 81116 expresses an LPS containing both lipo-oligosaccharides and O-antigen-like repeats, but nothing is known about the structure or sugar composition of these LPS species. A cosmid library of the genome of C. jejuni 81116 was constructed and probed with Campylobacter hyoilei genes involved in LPS synthesis. Five cosmids hybridized with the probe and two of these expressed C. jejuni 81116 LPS in Escherichia coli. By subcloning, a 16 kb DNA region was identified which contains the genetic information required to express C. jejuni LPS. DNA sequence analysis revealed 11 ORFs homologous to genes involved in LPS synthesis of other bacteria. They consisted of three homologues of sugar biosynthesis genes, two homologues of transport genes and six homologues of sugar transferases.