Sensitivity of staphylococcus epidermidis to chlorhexidine and associated resistance properties

Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

The endocrine basis for reproductive life-history trade-offs during the previtellogenic resting stage in the yellow fever mosquito, Aedes Aegypti

Juvenile hormone (JH) is the central hormonal regulator of life-history trade-offs in many insects. In Aedes aegypti, JH regulates reproductive development after emergence. Little is known about JH's physiological functions after reproductive development is complete or JH's role in mediating life-history trade-offs. By examining the effect of hormones, nutrition, and mating on ovarian physiology during the previtellogenic resting stage, critical roles were determined for these factors in mediating life-history trade-offs and reproductive output. The extent of follicular resorption during the previtellogenic resting stage is dependent on nutritional quality. Feeding females a low quality diet during the resting stage causes the rate of follicular resorption to increase and reproductive output to decrease. Conversely, feeding females a high quality diet causes resorption to remain low. The extent of resorption can be increased by separating the ovaries from a source of JH or decreased by exogenous application of methoprene. Active caspases were localized to resorbing follicles indicating that an apoptosis-like mechanism participates in follicular resorption. Accumulations of neutral lipids and the accumulation of mRNA's integral to endocytosis and oocyte development such as the vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) were also examined under various nutritional and hormonal conditions. The abundance of mRNA's and neutral lipid content increased within the previtellogenic ovary as mosquitoes were offered increasing sucrose concentrations or were treated with methoprene. These same nutritional and hormonal manipulations altered the extent of resorption after a blood meal indicating that the fate of follicles and overall fecundity depends, in part, on nutritional and hormonal status during the previtellogenic resting stage. Mating female mosquitoes also altered follicle quality and resorption similarly to nutrition or hormonal application and demonstrates that male accessory gland substances such as JH III passed to the female during copulation have a strong effect on ovarian physiology during the previtellogenic resting stage and can influence reproductive output. Taken together these results demonstrate that the previtellogenic resting stage is not an inactive period but is instead a period marked by extensive life-history and fitness trade-offs in response to nutrition, hormones and mating stimuli.

Discovery and Characterization of a Novel Fatty Acid Synthase Inhibitor with Antineoplastic Activity against Breast Cancer

During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.

From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.

Produção de biossurfactantes e nanoemulsões a partir da microalga spirulina

As microalgas têm sido foco de muitos estudos tendo em vista sua grande aplicabilidade na indústria de alimentos e farmacêutica, como também nas áreas da biomedicina e ambiental. A Spirulina é uma microalga que possui alto valor nutricional, apresenta alto teor proteico e é rica em substâncias bioativas. Esta microalga apresenta em sua composição compostos como glicolípidios, fosfolipídios e lipídios neutros, que por sua vez possuem efeito biossurfactante. Assim, o objetivo deste estudo foi verificar a potencialidade de produção de biossurfactantes a partir de diferentes cepas de Spirulina. Para isso, foram realizados experimentos utilizando Delineamento Fatorial Completo 22 , visando avaliar a influência da concentração de fósforo e nitrogênio no cultivo das microalgas Spirulina platensis Paracas, Spirulina platensis LEB 52 e Spirulina sp. LEB 18, como também nos extratos oriundos das microalgas, através da medida da tensão superficial. Foi também avaliada a influência destes nutrientes em extratos de Spirulina platensis LEB 52 e Spirulina sp. LEB 18 a partir do índice de emulsificação e diâmetro médio das gotículas das emulsões preparadas a partir dos extratos. Para extrações de biossurfactantes foram testados os solventes metanol, etanol e hexano. Nas formulações das nanoemulsões utilizou-se homogeneizador de alta velocidade, como fase aquosa os extratos oriundos das microalgas e como fase oleosa, óleo de girassol. As formulações foram preparadas utilizando-se diferentes concentrações da fase aquosa e oleosa, bem como diferentes velocidades e tempos de agitação. De acordo com os cultivos de Spirulina platensis Paracas realizados foi verificado que o cultivo que atingiu maior valor de concentração máxima de biomassa e maior produtividade foi realizado com 114 mg.L-1 de fósforo e sem adição de nitrogênio. Porém em relação às microalgas Spirulina platensis LEB 52 e Spirulina sp. LEB 18, as variáveis fósforo e nitrogênio não apresentaram influência significativa na concentração máxima de biomassa e produtividade máxima. O extrato que apresentou a menor tensão superficial (26,75 mN.m-1 ) foi verificado quando foi utilizado etanol como solvente, sendo este obtido a partir de cultivo da microalga Spirulina sp. LEB 18 realizado sem adição de nitrogênio e de fósforo. Em relação ao índice de emulsificação foram atingidos valores superiores a 59%, porém as concentrações utilizadas de nitrogênio e fósforo não apresentaram influência significativa nesta resposta. Neste trabalho foi possível obter nanoemulsões estáveis por até 30 d e com diâmetro médio de gotículas de até 532 nm. Os resultados obtidos neste trabalho são favoráveis à pesquisa na aplicação tanto dos extratos microalgais como das nanoemulsões obtidas apresentando potencialidade de uso em diversos processos industriais, como nas áreas ambiental, farmacêutica, cosmética e alimentos.

The periplasmic domain of the barrel assembly machinery protein A (BamA) from Escherichia coli assists folding of outer membrane protein A

The assembly of outer membranes of the cell wall of Gram-negative bacteria and of various organelles of eukaryotic cells requires the evolutionarily conserved β-barrel-assembly machinery (BAM) complex. This thesis describes the biochemical and biophysical properties of the periplasmic domain of the β-barrel assembly machinery protein A (PD-BamA) of the E. coli BAM complex, its effect on insertion and folding of the Outer membrane protein A (OmpA) into lipid bilayers and the identification of regions of PD-BamA that may be involved in protein-protein interactions. The secondary structure of PD-BamA in mixed lipid bilayers, analyzed by Circular dichroism (CD) spectroscopy, contained less β-sheet at an increased content of phosphatidylglycerol (PG) in the lipid membrane. This result showed membrane binding, albeit only in the presence of negatively charged lipids. Fluorescence spectroscopy demonstrated that PD-BamA only binds to lipid bilayers containing the negatively charged DOPG, confirming the results of CD spectroscopy. PD-BamA did not bind to zwitterionic but overall neutral lipid bilayers. PD-BamA bound to OmpA at a stoichiometry of 1:1. PD-BamA strongly facilitated insertion and folding of OmpA into lipid membranes. Kinetics of PD-BamA mediated folding of OmpA was well described by two parallel folding processes, a fast folding process and a slow folding process, differing by 2-3 orders of magnitude in their rate constants. The folding yields of OmpA depended on the concentration of lipid membranes and also on the lipid head groups. The presence of PD-BamA resulted in increased folding yields of OmpA in negatively charged DOPG, but PD-BamA did not affect the folding kinetics of OmpA into bilayers of zwitterionic but overall neutral lipids. The efficiency of folding and insertion of OmpA into lipid bilayers strongly depended on the ratio PD-BamA/OmpA and was optimal at equimolar concentrations of PD-BamA and OmpA. To examine complexes of unfolded OmpA with PD-BamA in more detail, site-directed spectroscopy was used to explore contact regions in both, PD-BamA and OmpA. Similarly, contact regions were also investigated for another protein complex formed by PD-BamA and the lipoprotein BamD. The obtained data suggest, that the site of interaction on PD-BamA for OmpA might be oriented towards the exterior environment away from the preceding POTRA domains, but that PD-BamA is oriented with its short α-helix α1 of POTRA domain 5 towards the C-terminal end of BamD.

Measurement of forces between and within bilayers of neutral phospholipids

Phospholipids in water form lamellar phases made up of alternating layers of water and bimolecular lipid leaflets. Three complementary methods, osmotic, mechanical, and vapour pressures, were used to measure the work of removing water from lamellar phases composed of frozen dipalmitoylphosphatidylcholine ( DPPC ), melted DPPC, egg phosphatidylethanolamine or equimolar mixtures of DPPC and cholesterol ( DPPC/CHOL ), Concurrently the structural changes that resulted from this water removal were measured using X-ray diffraction. The work was divided into that which forces the bilayers together ( F ) and that which compresses the molecules together within the bilayers ( F )# A large repulsive force exists between bilayers composed of each of the lipids studied and this force increases exponentially as bilayer separation is decreased. F is affected by the nature of the head groups, conformation of the acyl chains and heterogeneity of these chains. In general all of the melted phosphatidylcholines ( melted DPPC, egg lecithin and DPPC/CHOL ) have large equilibrium separations in excess water resulting from large repulsive hydration forces between these bilayers. By comparison, egg PE has an increased attractive force, and frozen DPPC has a decreased hydration force; each results in smaller separations in water for these two lipids. The chemical potentials of the water between the bilayers for all these lipids lie on a continuum, indicating that interbilayer water cannot be characterized by two discrete states, usually referred to as "bound" or "non**bound". For all lipids studied a maximum of 25 % of the total work done on the system goes into deforming the bilayers. The method used here viii to separate repulsion from deformation, developed for us by v. A. Parsegian, provides a unique method for the measurement of lateral pressure of a bilayer and its modulus of deformability ( Y ). Lateral pressure is affected by the nature of the head group, conformation and heterogeneity of the acyl chains. For small changes in molecular surface area ( A ) near equilibrium, both melted and frozen DPPC have similar values for the deformability modulus. Thus in this regime it requires about the same force to change the angle of tilt of frozen chains as it does to compress the fluid bilayer. The introduction of cholesterol into bilayers of DPPC reduces dramatically the lateral pressure of the bilayers over a large range of molecular surface areas ( A ). The variation in the magnitude of bilayer repulsion with different phospholipids provides a basis for the mechanism of lipid segregation in mixed lipid systems and suggests that interacting heterogeneous membranes may influence or modulate the composition of the opposing membrane. The measurements of deformabilities of bilayers provides a direct comparison of them with the properties of monolayers.

(Table 1) Concentrations of neutral and phenolic organohalogen compounds in adipose tissue, blood, brain and liver of polar bears (Ursus maritimus) from East Greenland

The tissue-specific composition of sum classes of brominated and chlorinated contaminants and metabolic/degradation byproducts was determined in adult male and female polar bears from East Greenland. Significantly (p < 0.05) higher concentrations of SUM-PCBs, various other organochlorines such as SUM-CHL, p,p'-DDE, SUM-CBz, SUM-HCHs, octachlorostyrene (OCS),SUM-mirex, dieldrin, the flame retardants SUM-PBDEs, and total-(R)-hexabromocyclododecane (HBCD), SUM-methylsulfonyl (MeSO2)-PCBs and 3-MeSO2-p,p'-DDE, were found in the adipose and liver tissues relative to whole blood and brain. In contrast, SUM-hydroxyl (OH)-PCB, 4-OH-heptachlorostyrene and SUM-OH-PBDE concentrations were significantly highest (p < 0.05) in whole blood, whereas the highest concentrations of SUM-OH-PBBs were found in the adipose tissue. Based on the total concentrations of all organohalogens in all three tissues and blood, the combined body burden was estimated to be 1.34 ± 0.12 g, where >91% of this amount was accounted for by the adipose tissue alone, followed by the liver, whole blood, and brain. These results show that factors such as protein association and lipid solubility appear to be differentially influencing the toxicokinetics, in terms of tissue composition/localization and burden, of organohalogen classes with respect to chemical structure and properties such as the type of halogenation (e.g., chlorination or bromination), and the presence or absence of additional phenyl group substituents (e.g., MeO and OH groups). The tissue- and blood-specific accumulation (or retention) among organohalogen classes indicates that exposure and any potential contaminant-mediated effects in these polar bears are likely tissue or blood specific.

Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells.

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.

(Table 1) Neutral and phenolic organohalogen compounds in blubber samples of ringed seals (Pusa hispida) from East Greenland

We report on the comparative bioaccumulation, biotransformation and/or biomagnification from East Greenland ringed seal (Pusa hispida) blubber to polar bear (Ursus maritimus) tissues (adipose, liver and brain) of various classes and congeners of persistent chlorinated and brominated contaminants and metabolic by-products: polychlorinated biphenyls (PCBs), chlordanes (CHLs), hydroxyl (OH-) and methylsulfonyl (MeSO2-) PCBs, polybrominated biphenyls (PBBs), OH-PBBs, polybrominated diphenyl ether (PBDE) and hexabromocyclododecane (HBCD) flame retardants and OH- and methoxyl (MeO-) PBDEs, 2,2-dichloro-bis(4-chlorophenyl)ethene (p,p'-DDE), 3-MeSO2-p,p'-DDE, pentachlorophenol (PCP) and 4-OH-heptachlorostyrene (4-OH-HpCS). We detected all of the investigated contaminants in ringed seal blubber with high frequency, the main diet of East Greenland bears, with the exception of OH-PCBs and 4-OH-HpCS, which indicated that these phenolic contaminants were likely of metabolic origin and formed in the bears from accumulated PCBs and octachlorostyrene (OCS), respectively, rather than being bioaccumulated from a seal blubber diet. For all of the detectable sum of classes or individual organohalogens, in general, the ringed seal to polar bear mean BMFs for SumPCBs, p,p'-DDE, SumCHLs, SumMeSO2-PCBs, 3-MeSO2-p,p'-DDE, PCP, SumPBDEs, total-(alpha)-HBCD, SumOH-PBDEs, SumMeO-PBDEs and SumOH-PBBs indicated that these organohalogens bioaccumulate, and in some cases there was tissue-specific biomagnification, e.g., BMFs for bear adipose and liver ranged from 2 to 570. The blood-brain barrier appeared to be effective in minimizing brain accumulation as BMFs were <= 1 in the brain, with the exception of SumOH-PBBs (mean BMF = 93±54). Unlike OH-PCB metabolites, OH-PBDEs in the bear tissues appeared to be mainly accumulated from the seal blubber rather than being metabolic formed from PBDEs in the bears. In vitro PBDE depletion assays using polar bear hepatic microsomes, wherein the rate of oxidative metabolism of PBDE congeners was very slow, supported the probability that accumulation from seals is the main source of OH-PBDEs in the bear tissues. Our findings demonstrated from ringed seal to polar bears that organohalogen biotransformation, bioaccumulation and/or biomagnification varied widely and depended on the contaminant in question. Our results show the increasing complexity of bioaccumulated and in some cases biomagnified, chlorinated and brominated contaminants and/or metabolites from the diet may be a contributing stress factor in the health of East Greenland polar bears.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.