The integration of lipid-sensing and anti-inflammatory effects: how the PPARs play a role in metabolic balance

The peroxisomal proliferating-activated receptors (PPARs) are lipid-sensing transcription factors that have a role in embryonic development, but are primarily known for modulating energy metabolism, lipid storage, and transport, as well as inflammation and wound healing. Currently, there is no consensus as to the overall combined function of PPARs and why they evolved. We hypothesize that the PPARs had to evolve to integrate lipid storage and burning with the ability to reduce oxidative stress, as energy storage is essential for survival and resistance to injury/infection, but the latter increases oxidative stress and may reduce median survival (functional longevity). In a sense, PPARs may be an evolutionary solution to something we call the 'hypoxia-lipid' conundrum, where the ability to store and burn fat is essential for survival, but is a 'double-edged sword', as fats are potentially highly toxic. Ways in which PPARs may reduce oxidative stress involve modulation of mitochondrial uncoupling protein (UCP) expression (thus reducing reactive oxygen species, ROS), optimising forkhead box class O factor (FOXO) activity (by improving whole body insulin sensitivity) and suppressing NFkB (at the transcriptional level). In light of this, we therefore postulate that inflammation-induced PPAR downregulation engenders many of the signs and symptoms of the metabolic syndrome, which shares many features with the acute phase response (APR) and is the opposite of the phenotype associated with calorie restriction and high FOXO activity. In genetically susceptible individuals (displaying the naturally mildly insulin resistant 'thrifty genotype'), suboptimal PPAR activity may follow an exaggerated but natural adipose tissue-related inflammatory signal induced by excessive calories and reduced physical activity, which normally couples energy storage with the ability to mount an immune response. This is further worsened when pancreatic decompensation occurs, resulting in gluco-oxidative stress and lipotoxicity, increased inflammatory insulin resistance and oxidative stress. Reactivating PPARs may restore a metabolic balance and help to adapt the phenotype to a modern lifestyle.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

A adição de sal à água tem sido utilizada para a mitigação de estresse e aumento da taxa de sobrevivência em peixes. O presente estudo avaliou o efeito do cloreto de sódio (0,0; 1,0; 3,0 e 6.0 g/l) nas concentrações de cortisol plasmático, glicemia, triglicerídios, proteínas total plasmática, hematócrito, hemoglobina, número de eritrócitos, glicogênio e lipídio hepáticos, e lipídio muscular em matrinxã Brycon amazonicum adultos após quatro horas de transporte e durante período de recuperação de 96 h. Amostras foram coletadas antes e depois do transporte, bem como 24 e 96 h após a chegada. O nível de cortisol plasmático estava mais elevado logo após o transporte quando comparado à condição inicial (pré-transporte), exceto para os peixes transportados com sal nas concentrações 3,0 e 6,0 g/l. Comportamento semelhante foi observado para a glicemia, porém os peixes dos tratamentos 0,0, 1,0 e 3,0 g/l necessitaram de período superior a 24 h para recuperar a condição inicial. Foram registrados níveis mais baixos de glicogênio hepático em peixes do tratamento controle (0,0 g/l). Os parâmetros hemoglobina, número de eritrócitos, proteína plasmática total e lipídio hepático não apresentaram alterações durante o período experimental. Os valores de hematócrito diminuíram logo após o transporte em todos os tratamentos, retornando aos níveis iniciais após 24 h. Todos os tratamentos apresentaram redução nos níveis de lipídio muscular e triglicerídios durante o período de recuperação. Os resultados sugerem que a adição de 6,0 g/l de sal na água de transporte reduz as alterações fisiológicas de estresse e que é necessário período de 96 h após o transporte para a recuperação da condição inicial de matrinxãs transportados sem a adição de sal.

Nanostructured Lipid Carriers as a Strategy to Improve the In Vitro Schistosomiasis Activity of Praziquantel

Praziquantel (PZQ) is a pyrazinoisoquinoline anthelmintic that was discovered in 1972 by Bayer Germany. Currently, due to its efficacy, PZQ is the drug of choice against all species of Schistosoma. Although widely used, PZQ exhibits low and erratic bioavailability because of its poor water solubility. Nanostructured lipid carriers (NLC), second-generation solid lipid nanoparticles, were developed in the 1990s to improve the bioavailability of poorly water soluble drugs. The aim of this study was to investigate nanostructured lipid carriers as a strategy to improve the efficacy. of PZQ in S. mansoni treatment. We prepared NLC2 and NLC4 by adding seventy percent glycerol monostearate (GMS) as the solid lipid, 30% oleic acid (OA) as the liquid lipid and two surfactant systems containing either soybean phosphatidylcholine/poloxamer (PC/P-407) or phosphatidylcholine/Tween 60 (PC/T60), respectively. The carriers were characterized by nuclear magnetic resonance, differential scanning calorimetry, thermogravimetric analysis and Fourier transform-infrared spectroscopy. The safety profile was evaluated using red cell hemolysis and in vitro cytotoxicity assays. The results showed that the encapsulation of PZQ in NLC2 or NLC4 improved the safety profile of the drug. Treatment efficacy was evaluated on the S. mansoni BH strain. PZQ-NLC2 and PZQ-NLC4 demonstrated an improved efficacy in comparison with free PZQ. The results showed that the intestinal transport of free PZQ and PZQ-NLC2 was similar. However, we observed that the concentration of PZQ absorbed was smaller when PZQ was loaded in NLC4. The difference between the amounts of absorbed PZQ could indicate that the presence of T60 in the nanoparticles (NLC4) increased the rigid lipid matrix, prolonging release of the drug. Both systems showed considerable in vitro activity against S. mansoni, suggesting that these systems may be a promising platform for the administration of PZQ for treating schistosomiasis.

Improvement of the physical performance is associated with activation of NO/PGC-l alpha/mtTFA signaling pathway and increased protein expressions of electron transport chain in gastrocnemius muscle from rats supplemented with L-arginine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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.

New synthetic strategies to tethered bilayer lipid membranes

Tethered bilayer lipid membranes provide an efficient, stable and versatile platform for the investigation of integrated membrane proteins. However, the incorporation of large proteins, as well as of proteins with a large submembrane part is still a very critical issue and therefore, further optimisation of the system is necessary. The central element of a tBLM is a lipid bilayer. Its proximal leaflet is, at least to some extend, covalently attached to a solid support via a spacer group. The anchor lipid consists of three distinct parts, a lipid headgroup, a spacer group and an anchor. All parts together influence the final bilayer properties. In the frame of this work, the synthesis of new thiolipids for tBLMs on gold has been investigated. The aim was to obtain molecules with longer spacers in order to increase the submembrane space. The systems obtained have been characterized using SPR and EIS. The results obtained during this study are multiple. First, the synthesis of a previously synthesized architecture was successfully scaled up in an industrial lab using a new synthetic approach. The synthesis of large amounts is now feasible. Then, the synthesis of the new thiolipids was carried out taking into account the following requirements: the increase of the submembrane space by having longer ethyleneglycol spacers, the attachment of the molecules to a gold substrate via a thiol bond, and the tunability of the lateral mobility by changing the lipid headgroup. Three different synthetic strategies have been investigated. The polymeric approach did not prove to be successful, merely because of the broad molecular weight distribution. The synthesis of heterofunctionally protected oligoethyleneglycols allowed to obtain ethyleneglycol moieties with 6 and 8 units, but the tedious purification steps gave very low yields. Finally, the block by block synthesis using ethyleneglycol precursors proved to be an efficient and fast method to synthesize the target molecules. Indeed, these were obtained with very high yields, and the separation was very efficient. A whole family of new compounds was obtained, having 6, 8 and 14 ethyleneglycol units and with mono- or diphytanyl lipid headgroups. This new pathway is a very promising synthetic strategy that can be used further in the development of new compounds of the tether system. The formation of bilayers was investigated for the different thiolipids mainly by using EIS. The electrical properties of a bilayer define the quality of the membrane and allow the study of the functionality of proteins embedded in such a system. Despite multiple trials to improve the system using self assembly, Langmuir Blodgett transfer, and detergent mixed vesicles, the new polymer thiolipids did not show as high electrical properties as tBLMs reported in the literature. Nevertheless, it was possible to show that a bilayer could be obtained for the different spacer lengths. These bilayers could be formed using self assembly for the first monolayer, and two different methods for bilayer formation, namely vesicle fusion and solvent exchange. We could furthermore show functional incorporation of the ion carrier valinomycin: the selective transport of K+ ions could be demonstrated. For DPHL, it was even possible to show the functional incorporation of the ion channel gramicidin. The influence of the spacer length is translated into an increase of the spacer capacitance, which could correspond to an increase in the capacity of charge accumulation in the submembrane space. The different systems need to be further optimised to improve the electrical properties of the bilayer. Moreover, the incorporation of larger proteins, and proteins bearing submembrane parts needs to be investigated.

From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

Protein-Protein- und Protein-Lipid-Wechselwirkungen beeinflussen die Oligomerisierung und Funktion des E. coli Aquaglyceroporins GlpF

Aquaporine sind hochselektive Transmembrankanäle, die in allen Lebensformen den Fluss von Wasser und kleinen, polaren Molekülen wie Glycerol über Lipidmembranen ermöglichen. Obwohl die Kanalpore für den Substratfluss im Monomer lokalisiert ist, liegen Aquaporine innerhalb biologischer Membranen als Homotetramere vor. Im Rahmen dieser Arbeit wurden proteinbezogene und lipidmembranassoziierte Einflüsse auf die Oligomerisierung und Funktion des bakteriellen Aquaglyceroporins GlpF sowohl in vitro als auch in vivo untersucht. rnDie erhöhte Stabilität der Aquaporinpore sowie Interaktion zwischen den GlpF-Monomeren sind Triebkräfte der Aquaporin-Tetramerisierung. Ferner erfordern die GlpF-Tetramerisierung und -Aktivität bei Abschirmung der Ladung anionischer Lipide und einer minimalen Membrandicke von 27 Å keine spezielle Lipidumgebung. Da anionische Lipide die GlpF-Funktion jedoch störten, kann die GlpF-Aktivität in vivo möglicherweise durch die selektive Anreicherung von anionischen Lipiden in der unmittelbaren Proteinumgebung reguliert werden. Ungünstige Lipid-GlpF-Interaktionen können jedoch in Lipidumgebungen mit hoher Ordnung in der Acylkettenregion entstehen, die zu einer Aggregation der GlpF-Tetramere und reduzierten Aktivität führen. rnFerner wurde die Auswirkung der nephrogenen Diabetes insipidus verursachenden Aquaporin 2-Punktmutation V71M auf die Oligomerisierung und Funktion des homologen, bakteriellen Aquaglyceroporins GlpF untersucht. Da weder die Oligomierisierung noch die Aktivität des homologen, bakteriellen Aquaglyceroporins eingeschränkt sind, beruht der Krankheitsmechanismus der Aquaporin 2-Mutante V71M vermutlich auf einem defekten Transportmechansimus im Menschen. rn

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.

PP141. The lipid transporters ABCA1 and ABCG1 are differentially expressed in preeclamptic and IUGR placentas

INTRODUCTION The ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 are highly expressed in the placenta in various compartments, including the villous syncytiotrophoblast (V-STB) and foetal endothelial cells. Among other not yet characterized functions, they play a role in the foeto-maternal transport of cholesterol and other lipophilic molecules. In humans, preliminary data suggest expressional changes of ABCA1 and ABCG1 in pathologic gestation, particularly under hypoxic conditions, but a systematic expression analysis in common human pregnancy diseases has never been performed. OBJECTIVES The aim of the present study was to characterize ABCA1 and ABCG1 expression in a large series of pathologic placentas, in particular from preeclampsia (PE) and intrauterine growth restriction (IUGR) which are associated with placental hypoxia. METHODS Placentas from 152 pathological pregnancies, including PE and/or HELLP (n=24) and IUGR (n=21), and 20 normal control placentas were assessed for their ABCA1 and ABCG1 mRNA and protein expression with quantitative RT-PCR and semi-quantitative immunohistochemical analysis, respectively. RESULTS ABCA1 protein expression in the V-STB was significantly less extensive in PE compared with normal controls (<10% of V-STB stained for ABCA1 in 58% PE placentas vs. 25% controls; p=0.035). Conversely, it was significantly more wide-spread in IUGR (>75% of V-STB stained in 57% IUGR placentas vs. 15% controls; p=0.009). Moreover, there was an insignificant trend for increased ABCA1 expression in fetal endothelial cells of stem villi in PE (p=0.0588). ABCA1 staining levels in V-STB were significantly associated with placental histopathological features related with hypoxia: they were decreased in placentas exhibiting syncytial knotting (p=0.033) and decidual vasculopathy (p=0.0437) and increased in low weight placentas (p=0.015). The significant and specific alterations in ABCA1 protein expression found at a specific cellular level were not paralleled by changes in ABCA1 mRNA abundance of total placental tissue. ABCG1 staining was universally extensive in the V-STB of normal placentas, always affecting more than 90% of V-STB surface. In comparison, ABCG1 staining of the V-STB was generally often reduced in pregnancy diseases. In particular, less than 90% of V-STB exhibited ABCG1 staining in 26% of PE placentas (p=0.022) and 35% of IUGR placentas (p=0.003). Similarly to ABCA1, ABCG1 mRNA expression in total placental tissue was not significantly different between controls and PE or IUGR. CONCLUSION ABCA1 and ABCG1 proteins are differentially expressed, with either down- or up-regulation, in the V-STB of placentas exhibiting features of chronic hypoxia, such as in PE and IUGR. This suggests that other factors in addition to hypoxia regulate the expression of placental lipid transporters. The specific changes on a cellular level were masked when only total tissue mRNA was analysed underlining the importance of cell specific expression analysis. The potential effects of decreased placental ABCA1 and ABCG1 expression on foetal nutrition and development remain to be elucidated.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Functions of lipid raft membrane microdomains at the blood-brain barrier

The blood-brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).

Cholesterol Transport and Regulation in the Mammary Gland.

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

Transport and uptake of immunogenic lipids

An increasing number of lipid mediators have been identified as key modulators of immunity. Among these is a family of glycolipids capable of cellular uptake, loading onto the MHC-like molecule CD1d and stimulation of NKT cells. NKT cells are particularly interesting because they bridge innate and adaptive immunity by coordinating the early events of dendritic cell maturation, recruitment of NK cells, CD4 and CD8 T cells, and B cells at the site of microbial injury. As such, their therapeutic manipulation could be of the greatest interest in vaccine design or active immunotherapy. However, the use of NKT cells as cellular adjuvant of immunity in the clinic will require a better knowledge of the pharmacology of lipid agonists in order to optimize their action and avoid potential unseen off-target effects. We have been studying extracellular transport and cellular uptake of NKT agonists for the past few years. This field is confronted to a very limited prior knowledge and a small set of usable tools. New technology must be put in place and adapted to answering basic immunology questions related to NKT cells. The intimate link between the pharmacology of glycolipids and lipid metabolism makes us believe that great variations of bioactivity could be seen in the general population when NKT agonists are used therapeutically.