Chimeric Antigen Receptor Immunotherapy for Solid Tumors: Choosing the Right Ingredients for the Perfect Recipe.

Chimeric antigen receptor T cell therapies are revolutionizing the clinical practice of hematological tumors, whereas minimal progresses have been achieved in the solid tumor arena. Multiple reasons have been ascribed to this slower pace: The higher heterogeneity, the hurdles of defining reliable tumor antigens to target, and the broad repertoire of immune escape strategies developed by solid tumors are considered among the major ones. Currently, several CAR therapies are being investigated in preclinical and early clinical trials against solid tumors differing in the type of construct, the cells that are engineered, and the additional signals included with the CAR constructs to overcome solid tumor barriers. Additionally, novel approaches in development aim at overcoming some of the limitations that emerged with the approved therapies, such as large-scale manufacturing, duration of manufacturing, and logistical issues. In this review, we analyze the advantages and challenges of the different approaches under development, balancing the scientific evidences supporting specific choices with the manufacturing and regulatory issues that are essential for their further clinical development.

Improving Provision of Care for Long-term Survivors of Lymphoma.

The progressive improvement of lymphoma therapies has led to a significant prolongation of patient survival and life expectancy. However, lymphoma survivors are at high risk of experiencing a range of early and late adverse effects associated with the extent of treatment exposure. Among these, second malignancies and cardiopulmonary diseases can be fatal, and neurocognitive dysfunction, endocrinopathy, muscle atrophy, and persistent fatigue can affect patients' quality of life for decades after treatment. Early recognition and reduction of risk factors and proper monitoring and treatment of these complications require well-defined follow-up criteria, close coordination among specialists of different disciplines, and a tailored model of survivorship care. We have summarized the major aspects of therapy-related effects in lymphoma patients, reviewed the current recommendations for follow-up protocols, and described a new hospital-based model of survivorship care provision from a recent multicenter Italian experience.

Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma.

BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.

Postprandial human triglyceride-rich lipoproteins increase chemoattractant protein secretion in human macrophages.

This study tested the hypothesis that postprandial triglyceride-rich lipoproteins (ppTGRL) have inflammatory effects in primary human monocyte-derived macrophages (HMDM). ppTGRL were isolated from normolipidemic human volunteers, and the production of chemokines and of inflammatory prostaglandins and leukotrienes via the arachidonic acid cascade in HMDM was determined, and their effect on monocyte chemotaxis were assessed. In addition, the possible role of extracellular lipases in the inflammatory effects of ppTGRL was evaluated. ppTGRL were found to increase the secretion of chemoattractants, including monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α and -1ß and IL-8, by HMDM and to have a stimulatory effect on monocyte chemotaxis. HMDM secretion of leukotrienes B4 (LTB4) and lipoxin A (LXA4), which are potent activators of monocyte migration, was also stimulated by ppTGRL. Inclusion of the lipoprotein lipase (LPL) inhibitor orlistat did not alter the effects of ppTGRL on chemokine production, and the expression of mRNA for LPL and other secreted lipases was unaffected by the lipoproteins. These findings support the hypothesis that ppTGRL induce the secretion of chemokines by macrophages which promote monocyte recruitment, and that extracellular lipolysis of the particles is not required for these effects and provide further evidence to indicate that the postprandial lipoproteins contribute to a pro-atherogenic pattern after a fat-rich meal.

Role of macrophage activation in the lipid metabolism of postprandial triacylglycerol-rich lipoproteins.

The potential link between the inflammatory effects of postprandial lipemia and the induction of macrophage foam cell formation by triacylglycerol-rich lipoproteins (TGRL) was studied using postprandial triacylglycerol-rich lipoproteins (ppTGRL) derived from human volunteers and primary human monocyte-derived macrophages (HMDM). Subjects were fed a test meal high in dairy fat, followed three hours later by isolation of serum ppTGRL. Pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes were induced in HMDM by treatment with lipopolysaccharide (LPS) or dexamethasone (DEX), respectively. ppTGRL caused a dose-dependent increase in both triacylglycerol (TG) and cholesterol (CH) accumulation in the cells. TG accumulation was unaffected by LPS or DEX treatment, but LPS as compared with DEX-treated HMDM were found to accumulate more CH, and this effect was greater than that induced by ppTGRL in untreated cells. LPS-treatment had no effect on lipid uptake from ppTGRL (via the LDLr, scavenger receptors or SR-B1) or on CH efflux, but the CH synthesis inhibitor mevinolin abolished the difference between CH accumulation in LPS-and DEX-treated cells, suggesting that CH synthesis is enhanced in the inflammatory state. Phospholipid (PL) synthesis was increased in inflammatory M1 as compared with anti-inflammatory M2 HMDM. Moreover, TG synthesis was decreased by ppTGRL in DEX-treated as compared with untreated cells. We conclude, therefore, inflammation causes a greater increase in the accumulation of neutral lipids than ppTGRL in macrophages, and that this effect is related to modulation of PL metabolism and possibly also CH synthesis. Thus, the inflammatory phenotype of macrophages influences their lipid metabolism, and is, therefore, likely to modulate the induction of macrophage lipid accumulation by lipoproteins associated with foam cell formation.

High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.

BACKGROUND: Hyperhomocysteinemia (HHcy) causes increased oxidative stress and is an independent risk factor for cardiovascular disease. Oxidative stress is now believed to be a major contributory factor in the development of non alcoholic fatty liver disease, the most common liver disorder worldwide. In this study, the changes which occur in homocysteine (Hcy) metabolism in high fat-diet induced non alcoholic fatty liver disease (NAFLD) in rats were investigated. METHODS AND RESULTS: After feeding rats a standard low fat diet (control) or a high fat diet (57% metabolisable energy as fat) for 18 weeks, the concentration of homocysteine in the plasma was significantly raised while that of cysteine was lowered in the high fat as compared to the control diet fed animals. The hepatic activities of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGS), the enzymes responsible for the breakdown of homocysteine to cysteine via the transsulphuration pathway in the liver, were also significantly reduced in the high fat-fed group. CONCLUSIONS: These results indicate that high fat diet-induced NAFLD in rats is associated with increased plasma Hcy levels caused by down-regulation of hepatic CBS and CGL activity. Thus, HHcy occurs at an early stage in high fat diet-induced NAFLD and is likely to contribute to the increased risk of cardiovascular disease associated with the condition.

Neutrophil unsaturated fatty acid release by GM-CSF is impaired in cystic fibrosis.

Dysregulated inflammation in cystic fibrosis (CF) is attributed to an altered production of inflammatory mediators derived from polyunsaturated lipids. In comparison to the arachidonic acid (AA) cascade, little is known about the modulation of docosahexaenoic acid (DHA) membrane release. We compared data on neutrophil DHA- and AA- release from both control (CT) and patients with CF using [3H]AA or [14C]DHA as a markers for, respectively, AA and DHA- release. Granulocyte-macrophage-colony stimulating factor stimulated DHA release from CT, but not CF, neutrophils. Comparison showed that both [14C]DHA and [3H]AA liberated after stimulation was higher in CT than in CF neutrophils. Since bioactive mediators derived from DHA are resolving factors and those derived from AA are both pro- and anti- inflammatory, these results suggest that CF is associated with a reduction of the release of PUFA-precursors of lipooxygenated resolving mediators. This leads to the hypothesis that defects in the resolving factors production could contribute to the inflammatory dysregulated processes in CF. Furthermore, the methodology used may help to improve knowledge on the regulation and resolution of inflammation.

Induction of non-alcoholic fatty liver disease and insulin resistance by feeding a high-fat diet in rats: does coenzyme Q monomethyl ether have a modulatory effect?

OBJECTIVE: The aim of this study was to investigate the development of non-alcoholic fatty liver disease (NAFLD) in response to a high-fat diet in rats and to test the hypothesis that dietary coenzyme Q monomethyl ether (CoQme) has antisteatogenic effects. METHODS: Rats were fed a standard low-fat diet (control) for 18 wk or a diet containing 35% fat (57% metabolizable energy) for 10 wk, then divided into three groups for the following 8 wk. One group was given CoQ9me (30mg/kg body weight per day in 0.3mL olive oil: high fat+CoQ9me), the second olive oil (0.3mL/d) only (high fat + olive oil), and the third group received no supplements (high fat). RESULTS: Insulin levels and the activity of alanine aminotransferase in the plasma were significantly increased in all high-fat diet groups, and the homeostasis model assessment of insulin resistance indicated insulin resistance. Triacylglycerol concentrations in whole plasma and in very low-density lipoprotein and low-density lipoprotein fractions were also raised. Liver histology showed lipid accumulation in animals fed the high-fat diets, and liver triacylglycerol levels were increased (2.5- to 3-fold) in all high-fat diet groups. These effects were not changed by the administration of CoQ9me. CONCLUSIONS: Rats fed a diet with 57% energy from fat showed insulin resistance, hypertriglyceridemia, increased very low-density lipoprotein production, hepatic steatosis, and liver damage, and thus provide a good model for the early stages of NAFLD. Dietary CoQ9me, however, did not ameliorate the damaging effects of the high-fat diet.

Neutrophil generation of inflammatory precursors is not modulated by docosahexaenoic acid.

OBJECTIVE AND DESIGN: It is believed that correction of membrane fatty acid deficiency in cystic fibrosis (CF) downregulates the synthesis of proinflammatory mediators. We tested the hypothesis that an increase of the proportion of docosahexaenoic acid (DHA) in the membrane in vitro changes the neutrophil response to Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: Treatment with DHA increased the secretion of interleukin(IL)-1|*alpha*| by CF neutrophils, but the secretion of other cytokines, CD11b expression, and arachidonic acid (AA) release were not affected either in CF or control (CT) neutrophils. Both with and without DHA, only one out of eight CF neutrophils responded to LPS with an increase of released AA, while five out of seven CT cells released more AA (CF vs. CT P < 0.05 by Fisher test). CONCLUSIONS: These results indicate that in neutrophils the beneficial effects of DHA on immune response are not directly related to the generation of proinflammatory precursors, and suggest that in CF the lower neutrophil AA generation in response to activation could cause insufficient production of lipid mediators involved in the resolution of lung inflammation.

Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages.

Macrophage lipid accumulation induced by low density lipoproteins (LDL) plays a pivotal role in atherosclerotic plaque development. Previous work showed that Ocimum basilicum extract, used as hypocholesterolemic agent by traditional medicine in Morocco, has hypolipidemic activity in rat acute hyperlipimidemia. This study investigated the effects of ethanolic extract of O. basilicum on lipid accumulation in human macrophages. As modification of LDL increase atherogenicity of the particles we evaluated the effects of the extract on LDL oxidation. The extract caused a dose-related increase of LDL-resistance to Cu(2+)-induced oxidation. Furthermore, at the dose of 60 microg/ml, significantly decreases the accumulation of macrophage lipid droplets induced by modified LDL evaluated as by red-oil staining. Cholesterol esterification and triacylglycerol synthesis in the cells were not affected. Macrophage treatment with 60 microg/ml, but not 20 microg/ml, of the extract reduced newly synthesized unesterified cholesterol by about 60% and decreased scavenger receptors activity by about 20-30%, evaluated by the internalization of cholesterol carried by [(3)H]CE-aggregated-LDL. The results suggest that O. basilicum ethanolic extract has the capability to reduce foam cell formation through the reduction of cholesterol synthesis and the modulation of the activity of surface scavenger receptors.

Effects of lycopene on the induction of foam cell formation by modified LDL.

The effect of lycopene on macrophage foam cell formation induced by modified low-density lipoprotein (LDL) was studied. Human monocyte-derived macrophages (HMDM) were incubated with lycopene in the presence or absence of native LDL (nLDL) or LDL modified by oxidation (oxLDL), aggregation (aggLDL), or acetylation (acLDL). The cholesterol content, lipid synthesis, scavenger receptor activity, and the secretion of inflammatory [interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha] and anti-inflammatory (IL-10) cytokines was determined. Lycopene was found to decrease the synthesis of cholesterol ester in incubations without LDL or with oxLDL while triacylglycerol synthesis was reduced in the presence of oxLDL and aggLDL. Scavenger receptor activity as assessed by the uptake of acLDL was decreased by approximately 30% by lycopene. In addition, lycopene inhibited IL-10 secretion by up to 74% regardless of the presence of nLDL or aggLDL but did not affect IL-1beta or TNF-alpha release. Lycopene also reduced the relative abundance of mRNA transcripts for scavenger receptor A (SR-A) in THP-1 macrophages treated with aggLDL. These findings suggest that lycopene may reduce macrophage foam cell formation induced by modified LDL by decreasing lipid synthesis and downregulating the activity and expression of SR-A. However, these effects are accompanied by impaired secretion of the anti-inflammatory cytokine IL-10, suggesting that lycopene may also exert a concomitant proinflammatory effect.

Effects of new combinative antioxidant FeAOX-6 and alpha-tocotrienol on macrophage atherogenesis-related functions.

Pivotal role in atherogenesis is played by macrophages, which are early site for lipid accumulation and mediate the inflammatory and immune response in the intima. Epidemiological evidence indicates that natural antioxidants reduce the risk of heart disease, but, so far, supplementation studies have failed to confirm any protective effects of these compounds against cardiovascular disease. This study evaluated the effects of the natural antioxidant alpha-tocotrienol and of the newly designed compound, FeAOX-6, which combines antioxidant structural features of both tocopherols and carotenoids into a single molecule, on macrophage functions involved in foam cell formation. FeAOX-6 or alpha-tocotrienol induce a strong dose-dependent reduction of cholesterol and reduce cholesterol accumulation in human macrophages. The extent of the reduction found with alpha-tocotrienol was greater than that induced by FeAOX-6 and did not correlate with their respective antioxidant capacities. Treatment of HMDM with alpha-tocotrienol or FeAOX-6 enhanced also tumor necrosis factor-alpha secretion. These results are consistent with a reduction in scavenger receptor activity, but we found that antioxidant treatment did not affect cholesterol uptake from modified LDL. The effects on release on pro-inflammatory prostanoid precursors, PGE(2) and cytokine suggest a variety of metabolic responses that are both dependent on antioxidant compounds and macrophages activation status.

Incorporation of lycopene into chylomicron remnant-like particles inhibits their uptake by HepG2 cells.

The influence of the incorporation of the antioxidant tomato pigment, lycopene, into chylomicron remnant-like particles (CRLPs) on their uptake by the liver cells was investigated. CRLPs or CRLPs containing lycopene (lycCRLPs) radiolabelled with [(3)H]triacylglycerol were incubated with cells of the human liver hepatoma cell line, HepG2, and the radioactivity taken up by the cells was determined. LycCRLPs were taken up significantly more slowly than CRLPs over a concentration range of 5-60 microg cholesterol/ml and a time course of 2-6 h. Pre-incubation of the hepatocytes with an excess of low density lipoprotein (LDL) inhibited the uptake of CRLPs by about 50%, but had no effect on the uptake of lycCRLPs, and under these conditions the CRLPs and lycCRLPs were taken up at similar rates. In HepG2 cells pre-treated with suramin, which inhibits uptake via the LDL receptor-related protein (LRP), the uptake of CRLPs was also inhibited (-37%) to a greater extent than that of lycCRLPs (-24%), so that the values for the two types of particle were no longer significantly different. Heparinase increased the uptake of lycCRLPs (about 2 fold), but not CRLPs, bringing it to a level equivalent to that seen with the control particles. These findings demonstrate that the incorporation of lycopene into CRLPs decreases their uptake by HepG2 cells and suggest that this effect is due to differential interaction with the LDL receptor and the LRP-receptor-mediated pathways, and may also involve binding of the particles to HSPG.

Very low density lipoprotein and low density lipoprotein isolated from patients with hepatitis C infection induce altered cellular lipid metabolism.

Several abnormalities of lipid metabolism, including hypo-beta-lipoproteinemia and liver steatosis are associated with infection by hepatitis C virus (HCV). The aim of this study was to determine whether circulating lipoproteins of patients with HCV infection could directly cause alterations of lipid cellular metabolism. To this end the metabolic response of human monocyte-derived macrophages (HMDM) to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), measuring the cholesteryl ester (CE) and triglyceride (TG) production was analyzed. Lipoproteins were isolated from 18 patients infected with hepatitis C virus (HCV-VLDL and HCV-LDL) and from normal healthy donors (ct-VLDL and ct-LDL). In comparison to ct-lipoproteins, HCV-lipoproteins induced significant differences in HMDM CE and TG production. HCV-VLDL decreased CE and TG production; while HCV-LDL induced an increased TG synthesis. The present findings suggest that HCV infection modifies VLDL and LDL molecular composition, affecting cellular lipid metabolism, thus promoting intracellular lipid accumulation and hypo-beta-lipoproteinemia.

Evidence of dual pathways for lipid uptake during chylomicron remnant-like particle processing by human macrophages.

BACKGROUND: Though it is now clear that chylomicron remnants are pro-atherogenic lipoproteins, events leading to their incorporation by macrophages are poorly understood. METHODS: This study investigates, in human macrophages, the fate of either [(3)H]cholesteryl oleate or [(3)H]triacylglycerol carried by human apolipoprotein-E-containing chylomicron remnant-like particles (CRLP) and the influence of CRLP containing trilinolein, (18:2)CRLP, or triolein, (18:1)CRLP, on lipid accumulation, newly synthesized cholesteryl ester (CE) and triacylglycerol (TG). RESULTS: Labelled fatty acids from TG were markedly incorporated into TG and phospholipid and, to a lesser extent, into free fatty acids and were scarcely recovered in cholesteryl esters. [(3)H]CE from CRLP accumulated in cells in a dose-dependent manner with a significant difference between concentrations of 10 and 40 microg cholesterol/ml with (18:2)CRLP. In the same concentration range, TG synthesis was enhanced by about 46 and 30% by (18:2)CRLP and (18:1)CRLP cholesterol, respectively, whereas the esterification of cholesterol, evaluated by [(3)H]oleate incorporation, was decreased by about 30% with both types of CRLP. Endocytosis inhibition did not prevent cell cholesterol and TG accumulation, whereas lipoprotein lipase inhibition reduced the TG content. CONCLUSIONS: The results are consistent with the hypotheses that in macrophages dietary remnants may support TG and CE internalization via different mechanisms. Extracellular lipolysis seems particularly important for internalization of dietary fatty acids, whereas the entrance of CE seems attributable to a concomitant selective CE uptake mediated by scavenger receptor class B type I, since the scavenger receptor class B type I antibody induces significant inhibition (38%) of [(3)H]CE transported by CRLP, but does not affect internalization of [(3)H]TG carried by the same particles.

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases.

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte-derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]-oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA-induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal-regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.

Protection of chylomicron remnants from oxidation by incorporation of probucol into the particles enhances their uptake by human macrophages and increases lipid accumulation in the cells.

The effects of protection of chylomicron remnants from oxidation on their uptake and induction of lipid accumulation in macrophages were investigated using chylomicron remnant-like particles (CRLPs) containing the lipophilic antioxidant drug, probucol, and macrophages derived from the human monocyte cell line, THP-1. The total lipid content of THP-1 macrophages was markedly higher (x2.2) after 48 h of incubation of THP-1 macrophages with CRLPs containing probucol (pCRLPs) when compared to CRLPs without probucol, and this was because of increases in triacylglycerol (x2.3) and cholesterol (x1.8) levels, while cholesteryl ester concentrations were not significantly changed. Determination of the uptake of CRLPs and pCRLPs by the cells using particles labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethylindo-carbocyanine perchlorate showed that pCRLPs are taken up at a faster rate than CRLPs. The synthesis of triacylglycerol, as measured by the incorporation of [(3)H]oleate and [(3)H]glycerol, was also increased in macrophages incubated with pCRLPs as compared to CRLPs without probucol, but phospholipid and cholesteryl ester formation from [(3)H]oleate was unaffected. In addition, no differences between the effects of CRLPs and pCRLPs on the expression of mRNA for a range of genes believed to be involved in lipoprotein uptake, intracellular lipid metabolism and the efflux of cholesterol from macrophages was detected. These results suggest that antioxidants carried in chylomicron remnants enhance lipid accumulation in macrophages by increasing the rate of uptake of the particles and raising the intracellular synthesis of triacylglycerol, but not cholesteryl ester, and that these effects are brought about by changes at the post-transcriptional level. Antioxidants carried in chylomicron remnants therefore may promote the development of atherosclerosis, and this is likely to be particularly important in conditions where clearance of remnants from the circulation is delayed.

Incorporation of lycopene into chylomicron remnant-like particles enhances their induction of lipid accumulation in macrophages.

Lipid accumulation in macrophages exposed to chylomicron remnant-like particles containing the dietary antioxidant lycopene was investigated. After incubation with THP-1 macrophages (48h), chylomicron remnant-like particles containing lycopene (lycCRLPs) as compared to those without (CRLPs) caused significantly more lipid accumulation in the cells, and this was due to increases in both the triacylglycerol (+100%) and cholesterol (+62%) content. In addition, expression of mRNA for diacylglycerol acyltransferase (DGAT), a key enzyme in triacylglycerol synthesis, was significantly decreased by lycCRLPs, but not CRLPs. These findings suggest that lycopene from the diet may promote, rather than retard, lipid accumulation in macrophages during its transport in the blood in chylomicron remnants.

Influence of thiol balance on micellar cholesterol handling by polarized Caco-2 intestinal cells.

The in vitro thiol redox modulation of cholesterol homeostasis was investigated in polarized Caco-2 intestinal cells. Cells were pre-incubated with the pro-oxidant compound CuSO4 or with the antioxidant N-acetylcysteine (NAC), to induce a mild shift of the intracellular redox potential toward, respectively, a more oxidizing or a more reducing equilibrium, via a manipulation of intracellular soluble thiols (glutathione). Then, monolayers were exposed to micellar cholesterol and both the cholesterol uptake and export, as well as the cholesteryl ester cycle, were analyzed. We found that pro-oxidizing conditions induced a significant cholesterol retention within the cells, particularly in the unesterified form (FC), as a result of an augmented sterol incorporation coupled with a reduced rate of FC esterification. A reduction in FC export was also evident. Furthermore, the combination of FC retention and the oxidative imbalance leads to significant alterations of the monolayer integrity, evidenced by both the enhanced tight junction permeability and the lactate dehydrogenase release into the basolateral medium. In contrast, a more reducing environment generated by NAC pre-treatment favors the limitation of the resident time of FC into the cells, via a reduced cholesterol uptake and a concomitant increased cholesterol esterification. In addition, a significant higher FC extrusion into the basolateral medium was also appreciable. Our results indicate that the thiol balance of intestinal cells may be critical for the regulation of cholesterol homeostasis at the intestinal level, influencing the lipid transport throughout the intestinal barrier.