Background: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. Objective: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. Methods: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. Results: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. Conclusion: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

Background: Lipid transfer proteins (LTPs) are stable and highly conserved proteins of around 10 kD. They have recently been identified as allergens in fruits of the Rosaceae family. Objective: The aim of this study was to investigate whether the highly conserved structure of LTPs justifies a designation as a true pan-allergen, and to study the role of protein stability in allergenicity. Methods: Thirty-eight patients with a positive skin prick test to Rosaceae fruit extracts were characterized by interviews and skin prick tests. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified natural and recombinant LTPs. To address the role of protein stability in the allergenicity of LTP, fruit extracts and LTPs were digested with pepsin. Results: IgE antibodies to Rosaceae LTPs cross-reacted with a broad range of non-Rosaceae vegetable foods. Inhibition studies with purified natural and recombinant LTPs confirmed the role of LTP in this cross-reactivity. Many of the patients with this type of cross-reactive IgE antibodies had a clinical food allergy. In contrast to the typical birch Rosaceae cross-reactive patients, the oral allergy syndrome was frequently accompanied by more severe and systemic reactions. IgE reactivity to LTP was shown to be resistant to pepsin treatment of the allergen. Conclusion: LTP is a true pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation. Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM. We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.

Objective-Advanced glycation end products (AGEs), such as N -(carboxymethyl)lysine (CML), are implicated in vascular disease. We previously reported increased CML accumulation in small intramyocardial blood vessels in diabetes patients. Diabetes patients have an increased risk for acute myocardial infarction (AMI). Here, we examined a putative relationship between CML and AMI. Methods and Results-Heart tissue was stained for CML, myeloperoxidase, and E-selectin in AMI patients (nϭ26), myocarditis patients (nϭ17), and control patients (nϭ15). In AMI patients, CML depositions were 3-fold increased compared with controls in the small intramyocardial blood vessels and predominantly colocalized with activated endothelium (E-selectin-positive) both in infarction and noninfarction areas. A trend of increased CML positivity of the intima of epicardial coronary arteries did not reach significance in AMI patients. In the rat heart AMI model, CML depositions were undetectable after 24 hours of reperfusion, but became clearly visible after 5 days of reperfusion. In line with an inflammatory contribution, human myocarditis was also accompanied by accumulation of CML on the endothelium of intramyocardial blood vessels. Conclusions-CML, present predominantly on activated endothelium in small intramyocardial blood vessels in patients with AMI, might reflect an increased risk for AMI rather than being a result of AMI.