Phospholipid chlorohydrin induces leukocyte adhesion to Apoe(-/-) mouse arteries via upregulation of P-selectin

HOCl-modified low-density lipoprotein (LDL) has proinflammatory effects, including induction of inflammatory cytokine production, leukocyte adhesion, and ROS generation, but the components responsible for these effects are not completely understood. HOCl and the myeloperoxidase-H2O2-halide system can modify both protein and lipid moieties of LDL and react with unsaturated phospholipids to form chlorohydrins. We investigated the proinflammatory effects of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine (SOPC) chlorohydrin on artery segments and spleen-derived leukocytes from ApoE-/- and C57 Bl/6 mice. Treatment of ApoE-/- artery segments with SOPC chlorohydrin, but not unmodified SOPC, caused increased leukocyte-arterial adhesion in a time- and concentration-dependent manner. This could be prevented by pretreatment of the artery with P-selectin or ICAM-1-blocking antibodies, but not anti-VCAM-1 antibody, and immunohistochemistry showed that P-selectin expression was upregulated. However, chlorohydrin treatment of leukocytes did not increase expression of adhesion molecules LFA-1 or PSGL-1, but caused increased release of ROS from PMA-stimulated leukocytes by a CD36-dependent mechanism. The SOPC chlorohydrin-induced adhesion and ROS generation could be abrogated by pretreatment of the ApoE-/- mice with pravastatin or a nitrated derivative, NCX 6550. These findings suggest that phospholipid chlorohydrins formed in HOCl-treated LDL could contribute to the proinflammatory effects observed for this modified lipoprotein in vitro.

Oxygen free radicals denature human IgG and increase its reactivity with rheumatoid factor antibody

Rheumatoid inflammation is characterised by the production of rheumatoid factor antibodies directed against denatured IgG. Oxygen free radicals have the potential to denature all manner of proteins and can be generated by activated phagocytic cells in the inflamed joint. By modifying routine ELISA and nephelometric procedures for measuring rheumatoid factor, (i.e. substituting free radical altered IgG for rabbit and heat aggregated IgG as antigens) we have observed that oxygen radicals, generated by (1) UV light and (2) PMA-activated neutrophils, give rise to monomeric and polymeric forms of IgG which have increased reactivity towards IgM and IgA polyclonal rheumatoid factor antibodies. We conclude that free radical alteration of IgG may be a stimulus to the formation of immune complexes with rheumatoid factor antibody, thereby promoting and amplifying tissue damage during rheumatoid inflammation.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

αα'-trehalose 6,6'-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro:simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swift recognition and removal of apoptotic cells is important for normal tissue homeostasis and failure in the underlying clearance mechanisms has pathological consequences associated with inflammatory and auto-immune diseases. Cell cultures in vitro usually lack the capacity for removal of non-viable cells because of the absence of phagocytes and, as such, fail to emulate the healthy in vivo micro-environment from which dead cells are absent. While a key objective in cell culture is to maintain viability at maximal levels, cell death is unavoidable and non-viable cells frequently contaminate cultures in significant numbers. Here we show that the presence of apoptotic cells in monoclonal antibody-producing hybridoma cultures has markedly detrimental effects on antibody productivity. Removal of apoptotic hybridoma cells by macrophages at the time of seeding resulted in 100% improved antibody productivity that was, surprisingly to us, most pronounced late on in the cultures. Furthermore, we were able to recapitulate this effect using novel super-paramagnetic Dead-Cert Nanoparticles to remove non-viable cells simply and effectively at culture seeding. These results (1) provide direct evidence that apoptotic cells have a profound influence on their non-phagocytic neighbors in culture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy for improving antibody manufacture in vitro.

Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFN? responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Novel tear film supplements:a potential application for synthetic protein-phospholipid complexes

Purpose: Surfactant proteins A, B, C and D complex with (phospho)lipids to produce surfactants which provide low interfacial tensions. It is likely that similar complexation occurs in the tear film and contributes to its low surface tension. Synthetic protein-phospholipid complexes, with styrene maleic anhydrides (SMAs) as the protein analogue, have been shown to have similarly low surface tensions. This study investigates the potential of modified SMAs and/or SMA-phospholipid complexes, which form under physiological conditions, to supplement natural tear film surfactants. Method: SMAs were modified to provide structural variants which can form complexes under varying conditions. Infrared spectroscopy and Nuclear Magnetic Resonance were used to confirm SMA structure. Interfacial behaviour of the SMA and SMA-phospholipid complexes was studied using Langmuir trough, du Nûoy ring and pulsating bubblemethods. Factors which affect SMA-phospholipid complex formation, such as temperature and pH, were also investigated. Results: Structural manipulation of SMAs allows control over complex formation, including under physiological conditions (e.g. partial SMAesterfication allowed complexation with dimyristoylphosphatidylcholine, at pH7). The low surface tensions of the SMAs (42mN/m for static (du Nûoy ring) and 34mN/m for dynamic (Langmuir) techniques) demonstrate their surface activity at the air-aqueous interface. SMA-phospholipid complexes provide even lower surface tensions (~2 mN/m), approaching that of lung surfactant, as measured by the pulsating bubblemethod. Conclusions: Design of the molecular architecture of SMAs allows control over their surfactant properties. These SMAs could be used as novel tear films supplements, either alone to complex with native tear film phospholipids or delivered as synthetic protein-phospholipid complexes.

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.

Polymer-phospholipid complexes for the solubilisation and delivery of drugs to the eye

Aim: Topical application of ophthalmic drugs is very inefficient; contact lenses used as drug delivery devices could minimize the drug loss and side effects. Styrene-maleic acid copolymers (PSMA) can form polymer-phospholipid complexes with dipalmitoyl phosphatidylcholine (DMPC) in the form of nanometric vesicles, which can easily solubilise hydrophobic drugs. They can be dispersed on very thin contact lens coatings to immobilize the drug on their surface. Methods: Two types of complexes stable at different pH values (5 and 7 respectively) where synthesized and loaded with drugs of different hydrophilicities during their formation process. The drug release was studied in vitro and compared to the free drug. Results: The mean sizes of the complexes obtained by light scattering were 50 nm and 450 nm respectively with low polydispersities. However, they were affected by the drugs load and release. An increase was observed in the duration of the release in the case of hydrophobic drugs, from days to weeks, avoiding initial “burst” and with a lesser amount of total drug released due to the interaction of the drug with the phospholipid core. The size and charge of the different drugs and the complexes nature also affected the release profile. Conclusions: Polymer-phospholipid complexes in the form of nanoparticles can be used to solubilise and release hydrophobic drugs in a controlled way. The drug load and release can be optimised to reach therapeutic values in the eye.

The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production

The use of cationic liposomes as experimental adjuvants for subunit peptide of protein vaccines is well documented. Recently the cationic liposome CAF01, composed of dimethyldioctadecylammonium (DDA) and trehalose dibehenate (TDB), has entered Phase I clinical trials for use in a tuberculosis (TB) vaccine. CAF01 liposomes are a heterogeneous population with a mean vesicle size of 500 nm; a strong retention of antigen at the injection site and a Th1-biassed immune response are noted. The purpose of this study was to investigate whether CAF01 liposomes of significantly different vesicle sizes exhibited altered pharmacokinetics in vivo and cellular uptake with activation in vitro. Furthermore, the immune response against the TB antigen Ag85B-ESAT-6 was followed when various sized CAF01 liposomes were used as vaccine adjuvants. The results showed no differences in vaccine (liposome or antigen) draining from the injection site, however, significant differences in the movement of liposomes to the popliteal lymph node were noted. Liposome uptake by THP-1 vitamin D3 stimulated macrophage-like cells did not show a liposome size-dependent pattern of uptake. Finally, whilst there were no significant differences in the IgG1/2 regardless of the liposome size used as a delivery vehicle for Ag85B-ESAT-6, vesicle size has a size dependent effect on cell proliferation and IL-10 production with larger liposomes (in excess of 2 µm) promoting the highest proliferation and lowest IL-10 responses, yet vesicles of ~500 nm promoting higher IFN-? cytokine production from splenocytes and higher IL-1ß at the site of injection.

Antibody detection of Burkholderia pseudommallei and Burkholderia mallei

Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.

The histogenesis of lymphoid organs in Tilapia mossambica and antibody responses in adults and fry

The thymic anlagen appears in Tilapia mossambica at 2 days post hatching and becomes lymphoid at 5 days. Lymphoid cells were first seen in the pronephros at 14 days and in the spleen at approximately five weeks of age. Differentiation into red and white pulp regions was seen by 10 weeks of age. Light and electron microscopic studies of adult lymphoid organ revealed increases in size and lymphoid cell numbers. Adult thymus develops a clearer corticomedullary differentiation of thymic corpuscles in the medulla and in the splenic red and white pulp became more distinct. Melanomacrophage centres were seen in spleen and pronephros. Adult fish gave primary and secondary antibody responses following challenge with sheep red bloods cells (SRBC), Escherichia coli (E. coli) and human gamma globulin (HGG). Plaque forming cell and immunocytoadherence assays revealed that head kidney and spleen were major sites for antibody production and development of antigen reactive cells. Proliferative activity in these organs was revealed using autoradiography and scintillation counting. Increased levels of pyroninophilia were also seen following antigenic challenge. Pilot studies on adults revealed that they were capable of rejecting first and second set allografts and leucocytes from spleen and head kidney proliferated in mixed leucocyte cultures. Antibody responses to SRBC, E. coli and HGG develop at about 10-12 weeks of age. Fry given either a single injection of SRBC at 10 weeks or two injections of the same antigen at 10 weeks and 12 days later, failed to respond to a further challenge with SRBC 56 days after the first injection (A time when animals would normally respond positively to this antigen). Injection of E. coli at the same times resulted in a prolonged antibody response.

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4 roles for cd14, lps-binding protein, and md2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor- production, IB degradation, p38 MAPK phosphorylation, and NF-B-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from 30 µM. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Chemistry of phospholipid oxidation

The oxidation of lipids has long been a topic of interest in biological and food sciences, and the fundamental principles of non-enzymatic free radical attack on phospholipids are well established, although questions about detail of the mechanisms remain. The number of end products that are formed following the initiation of phospholipid peroxidation is large, and is continually growing as new structures of oxidized phospholipids are elucidated. Common products are phospholipids with esterified isoprostane-like structures and chain-shortened products containing hydroxy, carbonyl or carboxylic acid groups; the carbonyl-containing compounds are reactive and readily form adducts with proteins and other biomolecules. Phospholipids can also be attacked by reactive nitrogen and chlorine species, further expanding the range of products to nitrated and chlorinated phospholipids. Key to understanding the mechanisms of oxidation is the development of advanced and sensitive technologies that enable structural elucidation. Tandem mass spectrometry has proved invaluable in this respect and is generally the method of choice for structural work. A number of studies have investigated whether individual oxidized phospholipid products occur in vivo, and mass spectrometry techniques have been instrumental in detecting a variety of oxidation products in biological samples such as atherosclerotic plaque material, brain tissue, intestinal tissue and plasma, although relatively few have achieved an absolute quantitative analysis. The levels of oxidized phospholipids in vivo is a critical question, as there is now substantial evidence that many of these compounds are bioactive and could contribute to pathology. The challenges for the future will be to adopt lipidomic approaches to map the profile of oxidized phospholipid formation in different biological conditions, and relate this to their effects in vivo. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.