Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Effects of margarines and butter consumption on lipid profiles, inflammation markers and lipid transfer to HDL particles in free-living subjects with the metabolic syndrome

Objective: Our purpose was to examine the effects of daily servings of butter, no-trans-fat margarine and plant sterol margarine, within recommended amounts, on plasma lipids, apolipoproteins (Apos), biomarkers of inflammation and endothelial dysfunction, and on the transfer of lipids to HDL particles in free-living subjects with the metabolic syndrome. Methods: This was a randomized, single-blind study where 53 metabolic syndrome subjects (62% women, mean age 54 years) received isocaloric servings of butter, no-trans-fat margarine or plant sterol margarine in addition to their usual diets for 5 weeks. The main outcome measures were plasma lipids, Apo, inflammatory and endothelial dysfunction markers (CRP, IL-6, CD40L or E-selectin), small dense LDL cholesterol concentrations and in vitro radioactive lipid transfer from cholesterol-rich emulsions to HDL. Difference among groups was evaluated by analysis of variance. Results: There was a significant reduction in Apo-B (-10.4 %, P = 0.043) and in the Apo-B/Apo-A-1 ratio (-11.1%, P = 0.034) with plant sterol margarine. No changes in plasma lipids were noticed with butter and no-trans-fat margarine. Transfer rates of lipids to HDL were reduced in the no-trans-fat margarine group: triglycerides -42.0%, (P<0.001 vs butter and sterol margarine) and free cholesterol -16.2% (P = 0.006 vs sterol margarine). No significant effects were noted on the concentrations of inflammatory and endothelial dysfunction markers among the groups. Conclusions: In free-living subjects with the metabolic syndrome consumption of plant sterol and no-trans-fat margarines within recommended amounts reduced, respectively, Apo-B concentrations and the ability of HDL to accept lipids. European Journal of Clinical Nutrition (2010) 64, 1141-1149; doi:10.1038/ejcn.2010.122; published online 21 July 2010

Orange juice decreases low-density lipoprotein cholesterol in hypercholesterolemic subjects and improves lipid transfer to high-density lipoprotein in normal and hypercholesterolemic subjects

Orange juice (OJ) is regularly consumed worldwide, but its effects on plasma lipids have rarely been explored. This study hypothesized that consumption of OJ concentrate would improve lipid levels and lipid metabolism, which are important in high-density lipoprotein (HDL) function in normolipidemic (NC) and hypercholesterolemic (HCH) subjects. Fourteen HCH and 31 NC adults consumed 750 mL/day OJ concentrate (1:6 OJ/water) for 60 days. Eight control subjects did not consume OJ for 60 days. Plasma was collected before and on the last clay for biochemical analysis and an in vitro as

Metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein in young obese and normal-weight patients with polycystic ovary syndrome

Objective: To clarify whether the metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein (HDL) are altered in patients with polycystic ovary syndrome (PCOS). Design: Case control study. Setting: Endocrinology clinics. Patient(s): Eight normal-weight (NW) and 15 obese (013) patients with PCOS were compared with 10 NW and 10 Ob women without PCOS paired for age and body mass index. Intervention(s): Determination of triglyceride-rich lipoprotein metabolism and lipid transfer to HDL. Main Outcome Measure(s): Participants were injected triglyceride-rich emulsions labeled with (14)C-cholesteryl esters and (3)H-triglycerides and the fractional clearance rate (FCR, in min(-1)) of labels was determined. Lipid transfer from artificial nanoemulsions to HDL was performed by incubating radioactively labeled lipid nanoemulsions with plasma during 1 hour, followed by radioactive counting of HDL-containing supernatant after chemical precipitation. Result(s): Lipolysis estimated by triglyceride FCR was equal in PCOS groups (NW = 0.043 +/- 0.032, Ob = 0.033 +/- 0.009) and respective controls (NW = 0.039 +/- 0.015, Ob = 0.044 +/- 0.019). However, the remnant removal as estimated by cholesteryl ester FCR was reduced in both PCOS groups (NW = 0.005 +/- 0.006, Ob = 0.005 +/- 0.005) compared with controls (NW = 0.016 +/- 0.006, Ob = 0.011 +/- 0.072). Lipid transfer rates were not different among groups, but triglyceride transfer rates were positively correlated with homeostasis model assessment estimate of insulin resistance in PCOS. Conclusion(s): PCOS patients showed decreased removal of atherogenic remnants even when fasting glucose was <100 mg/dL. This reinforces the usefulness of the measures taken to prevent cardiovascular events in PCOS patients. (Fertil Steril (R) 2010;93:1948-56. (C)2010 by American Society for Reproductive Medicine.)

HDL concentration, lipid transfer to HDL, and HDL size in normolipidemic nonobese menopausal women

Objective: To determine the impact of menopause on lipid transfer from donor lipoproteins to high-density lipoproteins (HDLs)-a process that is related to the protective function of HDL-and the size of HDL particles. Method: Plasma from 22 prernenopausal and 18 postmenopausal nonobese, normolipidemic women paired for age (40-50 years) was incubated in an artificial nanoemulsion labeled with radioactive lipids. Then the HDL fraction was assessed for radioactivity; the percentage of radioactive lipids transferred from the nanoemulsion to HDL was determined; and the size of HDL particles was measured by laser light scattering. Results: There were no differences between the 2 groups in serum concentration of HDL cholesterol (61 12 mg/dL vs 61 +/- 14 mg/dL) or apolipoprotein A(1) (1.5 +/- 0.3 g/L vs 1.5 +/- 0.2 g/L); lipid transfer to HDL; or size of HDL particles (8.8 +/- 0.8 vs 9.0 +/- 0.5 nm). Conclusion: Menopause was not found to affect HDL cholesterol plasma concentration, lipid transfer to HDL, or size of HDL particles in normolipidemic nonobese women. (C) 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd.All rights reserved.

Alterations in lipid transfer to High-Density Lipoprotein (HDL) and activity of paraoxonase-1 in HIV+ patients

HIV+ patients often develop alterations of the plasma lipids that may implicate in development of premature coronary artery disease. High-density lipoprotein (HDL) has an important role in preventing atherogenesis and the aim of this study was to investigate aspects of HDL function in HIV+ patients. HIV+ patients (n = 48) and healthy control subjects (n = 45) of both sexes with similar age were studied. Twenty-five were not being treated with antiretroviral agents, 13 were under reverse transcriptase inhibitor nucleosidic and non-nucleosidic (NRTI+NNRTI) and 10 were under NRTI + protease inhibitors (NRTI+PI) treatment. Paraoxonase 1 (PON1) activity and the transfer of free and esterified cholesterol, tryglicerides and phospholipids from a lipidic nanoemulsion to HDL were analyzed. In comparison with healthy controls, HIV+ patients presented low PON-1 activity and diminished transfer of free cholesterol and tryglicerides. In contrast, phospholipid transfer was increased in those patients, whereas the transfer of cholesteryl esters was unchanged. NRTI+NNRTI increases the transfer of cholesteryl esters and triglycerides but in NRTI+PI there was no difference in respect to non-treated HIV+ patients. HDL from HIV+ patients has smaller antioxidant properties, as shown by lower PON-1 activity, and the transfer of lipids to this lipoprotein fraction is also altered, suggesting that HDL function is defective in those patients.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Orange juice decreases low-density lipoprotein cholesterol in hypercholesterolemic subjects and improves lipid transfer to high-density lipoprotein in normal and hypercholesterolemic subjects

Orange juice (OJ) is regularly consumed worldwide, but its effects on plasma lipids have rarely been explored. This study hypothesized that consumption of OJ concentrate would improve lipid levels and lipid metabolism, which are important in high-density lipoprotein (HDL) function in normolipidemic (NC) and hypercholesterolemic (HCH) subjects. Fourteen HCH and 31 NC adults consumed 750 mL/day OJ concentrate (1:6 OJ/water) for 60 days. Eight control subjects did not consume OJ for 60 days. Plasma was collected before and on the last clay for biochemical analysis and an in vitro as

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with C-14-cholesteryl ester and H-3-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14 C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the H-3-free- cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

Lipid Transfer to HDL is Higher in Marathon Runners than in Sedentary Subjects, but is Acutely Inhibited During the Run

Although exercise increases HDL-cholesterol, exercise-induced changes in HDL metabolism have been little explored. Lipid transfer to HDL is essential for HDL's role in reverse cholesterol transport. We investigated the effects of acute exhaustive exercise on lipid transfer to HDL. We compared plasma lipid, apolipoprotein and cytokine levels and in vitro transfer of four lipids from a radioactively labeled lipid donor nanoemulsion to HDL in sedentary individuals (n = 28) and in marathon runners (n = 14) at baseline, immediately after and 72 h after a marathon. While HDL-cholesterol concentrations and apo A1 levels were higher in marathon runners, LDL-cholesterol, apo B and triacylglycerol levels were similar in both groups. Transfers of non-esterified cholesterol [6.8 (5.7-7.2) vs. 5.2 (4.5-6), p = 0.001], phospholipids [21.7 (20.4-22.2) vs. 8.2 (7.7-8.9), p = 0.0001] and triacylglycerol [3.7 (3.1-4) vs. 1.3 (0.8-1.7), p = 0.0001] were higher in marathon runners, but esterified-cholesterol transfer was similar. Immediately after the marathon, LDL- and HDL-cholesterol concentrations and apo A1 levels were unchanged, but apo B and triacylglycerol levels increased. Lipid transfer of non-esterified cholesterol [6.8 (5.7-7.2) vs. 5.8 (4.9-6.6), p = 0.0001], phospholipids [21.7 (20.4-22.2) vs. 19.1 (18.6-19.3), p = 0.0001], esterified-cholesterol [3.2 (2.2-3.8) vs. 2.3 (2-2.9), p = 0.02] and triacylglycerol [3.7 (3.1-4) vs. 2.6 (2.1-2.8), p = 0.0001] to HDL were all reduced immediately after the marathon but returned to baseline 72 h later. Running a marathon increased IL-6 and TNF-alpha levels, but after 72 h these values returned to baseline. Lipid transfer, except esterified-cholesterol transfer, was higher in marathon runners than in sedentary individuals, but the marathon itself acutely inhibited lipid transfer. In light of these novel observations, further study is required to clarify how these metabolic changes can influence HDL composition and anti-atherogenic function.

Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.

Abstract Background Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats. Results In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed. Conclusions It is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with 14C-cholesteryl ester and ³H-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the ³H-free-cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^