Parental experience of a risky environment leads to improved offspring growth rate.

Parasites (or diseases) are a major selective force for the evolution of life history traits and parasite-host evolution. Mothers can show a variety of responses to parasites during pregnancy, with different consequences for them or their offspring. However, whether information in the maternal environment before pregnancy can cause a change in the phenotype of the offspring is unknown. To avoid the confounding effect of pathogens and to reduce the risk of a direct effect of maternal immune system activation, we injected female laboratory mice with lipopolysaccharides (LPS) before they mated. In order to provide constant information on the potential infectious risk of the environment, females were mated with males that were also exposed to LPS before mating. Offspring from immune-challenged parents were larger and grew at a faster rate than offspring from control parents (injected with PBS). Additionally, offspring from immune-challenged parents that suffered the most from inflammation grew at a faster rate than offspring from low suffering parents. Producing heavier offspring that will reach sexual maturity earlier is likely to have fitness benefits for parents and offspring through improved reproductive success.

Phase I study of OM-174, a lipid A analogue, with assessment of immunological response, in patients with refractory solid tumors.

BACKGROUND: Lipids A, the lipophilic partial structure of lipopolysaccharides, induce regression of several tumor types in animal models. Rather than exerting direct cytotoxic effect, these compounds trigger the immune system which in turn stimulates secretion of cytokines, and activates the inducible nitric oxide synthase, as well as immune cell infiltration of tumors. OM-174 is an analogue of lipid A with dual action on Toll-like receptors 2 and 4. In an experimental model of peritoneal carcinomatosis induced in BDIX rats by intraperitoneal injection of syngeneic PROb colon cancer cells, it induced a complete regression of tumors. The present phase I trial was conducted to determine the maximum tolerated dose, the recommended phase II dose and biological response associated with OM-174 administered as intravenous infusion. METHODS: Patients received OM-174 twice weekly for a total of 5, 10 or 15 injections of either 600, 800 or 1000 µg/m(2). Blood samples for pharmacokinetic analysis and cytokine dosages were collected. NK cells activity and Toll-like receptors 4 polymorphism analysis were also performed. RESULTS: Seventeen patients were included. The highest dose administered was 1000 µg/m(2) repeated in 15 injections. The most common toxicities were a chills, fever, nausea/vomiting, diarrhea, fatigue and headache. No patient experienced haematological side effects. As no dose limiting toxicity was observed, despite a grade 3 respiratory complication, the maximal tolerated dose and recommended dose were not established. Three patients exhibited disease stabilization with a mean duration of 4 months. Pharmacokinetic profile of OM-174 was characterized by a low distribution volume and clearance. Analysis of TLR 4 polymorphysm showed that most (16/17) patients carried the wild type alleles. A progressive increase in NK cell number and activity was observed only in patients receiving 1000 µg/m(2) of OM-174. A peak of IL-8 and IL-10 concentrations were observed after each OM-174 injection. Peaks of TNF-alpha and IL-6 concentrations were detected after the first infusion and decreased progressively suggesting tolerance. CONCLUSION: OM-174 therapy was well tolerated at biologically active concentrations. Whereas the recommended dose was not determined, further studies are planned in combination with chemotherapy as animal models suggest a strong synergistic antitumor effect. TRIAL REGISTRATION: NCT01800812 (ClinicalTrials.gov Identifier).

Performance evaluation of human cytokines profiles obtained by various multiplexed-based technologies underlines a need for standardization.

BACKGROUND: Multiplexed methods permit simultaneous quantification of multiple cytokines. As several manufacturers offer reagents to quantify the same cytokines on a single instrument, comparison of the distribution should be made to determine whether these data are comparable from one assay to another. METHODS: We performed the quantification of cytokines in serum samples with three commercially available assays: Cytometric Bead Array (CBA), Protein Biochip Array Technology (PBAT), and Luminex Technology analysis. Using detection limit and reference range of the three commercial multiplex technologies, we evaluated: 1) the overall distribution of cytokines; and 2) the clinical impact. RESULTS: The three cytokines, IL-1ß, IL-1α and IL-4, cannot be measured by these methods because of the high number of non-detected data (>50%). By contrast, four cytokines as IL-8, VEGF, MCP-1 and EGF exhibited a low percentage of non-detected data whatever method was used. The comparison of the percentage of samples with values higher than the respective reference range of each method reported an absence of clinical concordance (Cohen's κ-test <0.40). CONCLUSIONS: Our results highlight the lack of transferability between the three commercially available multiplex methods evaluated (CBA, PBAT and Luminex Technology). Analytical performances are adequate for longitudinal studies using a same methodology but caution should be used for comparisons between results obtained with different methods underlying a need for standardization.

Glycogen synthase kinase 3 involvement in the excessive proinflammatory response to LPS in patients with decompensated cirrhosis.

BACKGROUND & AIMS: In decompensated cirrhosis, the early innate immune response to the Toll-like receptor 4 (TLR4) agonist, lipopolysaccharides (LPS), is characterized by a hyper-production of pro-inflammatory cytokines and hypo-production of the anti-inflammatory cytokine IL-10. In LPS-stimulated non-cirrhotic immune cells, the constitutively active glycogen synthase kinase (GSK) 3 favors pro- vs. anti-inflammatory cytokines, by acting on gene induction. However, in these cells, TLR4 dampens its own pro-inflammatory response by inducing early (within minutes) AKT-mediated phosphorylation of GSK3ß (one of two GSK3 isoforms) on Ser9. Phosphorylation of GSK3ß (Ser9) inhibits its activity, decreases pro-inflammatory cytokines, and increases IL-10. Thus, we investigated the role of GSK3 in LPS-induced cytokine production by peripheral blood mononuclear cells (PBMCs) or monocytes from patients with advanced cirrhosis and normal subjects. METHODS: Cells were pre-incubated with or without GSK3 inhibitor (SB216763 or lithium chloride) for 1h and then stimulated with LPS. Cytokine production was assessed at mRNA and secreted proteins levels, by real-time RT-PCR at 1h and ELISA at 20 h, respectively. GSK3ß phosphorylation was assessed using Western blotting. RESULTS: In cirrhotic and normal PBMCs pretreated with GSK3 inhibitors, LPS-induced production of pro-inflammatory proteins TNF-α and IL-12p40 was significantly decreased while that of IL-10 was increased. LPS-induced, AKT-mediated phosphorylation of GSK3ß on Ser9 found in normal monocytes, was abolished in cirrhotic cells. CONCLUSIONS: GSK3 is involved in the early TLR4-mediated pro-inflammatory response in patients with decompensated cirrhosis. This was associated with a defect in AKT-mediated GSK3ß phosphorylation resulting in unrestricted 'pro-inflammatory' activity of the enzyme.

Impact of 7-ketocholesterol and very long chain fatty acids on oligodendrocyte lipid membrane organization: evaluation via LAURDAN and FAMIS spectral image analysis.

In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 µM), C24:0 and C26:0 (10, 20 µM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 µM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 µM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 µM), C24:0 and C26:0 (20 µM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 µM) is more efficient than C24:0 and C26:0 (20 µM), to trigger lateral lipid membrane disorganization.

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes.

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l-peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1, Abcd2, Abcd3, and Acox1) involved in peroxisomal transport or beta-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l-peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.

7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder induced by a mutation in the ABCD1 gene, which causes the accumulation of very long-chain fatty acids in tissue and plasma. Oxidative stress may be a hallmark of X-ALD. In the plasma of X-ALD patients with different forms of the disease, characterized by high levels of C24:0 and C26:0, we observed the presence of oxidative stress revealed by decreased levels of GSH, α-tocopherol, and docosahexaenoic acid (DHA). We showed that oxidative stress caused the oxidation of cholesterol and linoleic acid, leading to the formation of cholesterol oxide derivatives oxidized at C7 (7-ketocholesterol (7KC), 7ß-hydroxycholesterol (7ß-OHC), and 7α-hydroxycholesrol (7α-OHC)) and of 9- and 13-hydroxyoctadecadienoic acids (9-HODE, 13-HODE), respectively. High levels of 7KC, 7ß-OHC, 7α-OHC, 9-HODE and 13-HODE were found. As 7KC induces oxidative stress, inflammation and cell death, which could play key roles in the development of X-ALD, the impact of 7KC on the peroxisomal status was determined in microglial BV-2 cells. Indeed, environmental stress factors such as 7KC could exacerbate peroxisomal dysfunctions in microglial cells and thus determine the progression of the disease. 7KC induces oxiapoptophagy in BV-2 cells: overproduction of H2O2 and O2-, presence of cleaved caspase-3 and PARP, nuclear condensation and/or fragmentation; elevated [LC3-II/LC3-I] ratio, increased p62 levels. 7KC also induces several peroxisomal modifications: decreased Abcd1, Abcd2, Abcd3, Acox1 and/or Mfp2 mRNA and protein levels, increased catalase activity and decreased Acox1-activity. However, the Pex14 level was unchanged. It is suggested that high levels of 7KC in X-ALD patients could foster generalized peroxisomal dysfunction in microglial cells, which could in turn intensify brain damage.

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes.

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7ß-hydroxycholesterol (7ßOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7ßOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7ßOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7ßOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains.

Correlational selection on pro- and anti-inflammatory effectors.

Parasites impose a permanent threat for hosts. As a consequence, immune defenses are important for host fitness. However, the immune response can also produce self-damage and impair host fitness if not properly regulated. Effectors that up- and downregulate the immune response should, therefore, evolve in concert, and be under the action of correlational selection. To address this issue, we assessed the shape of the selection operating on pro- and anti-inflammatory effectors following an inflammatory challenge in laboratory mice.We found that selection acts on the combination of these two traits as individuals that produced large amount of pro-inflammatory cytokines could achieve relatively high fitness (survival) only if also producing a large amount of anti-inflammatory effectors. To our knowledge, this is the first study providing evidence for correlational selection on immunity.

A moderate red wine intake improves blood lipid parameters and erythrocytes membrane fluidity in post myocardial infarct patients.

While the cardioprotective effect of moderate and regular wine consumption in primary prevention has been well documented, the goal of the present investigation was to explore the effect of wine intake on blood parameters (lipid, anti-oxidant capacity, and erythrocyte membrane potential and fluidity) in post myocardial infarct patients to evaluate perspectives in secondary prevention. A clinical intervention trial has been undertaken on a group of selected post myocardial infarct patients who gave written informed consent for participation in this study prior to enrolment. This two-week study has been conducted on hospitalized patients during a cardiac readaptation period. During this period, patients were submitted to a "Western prudent" diet (inspired by the Mediterranean diet) and two groups have been compared on a drawn basis: patients receiving red wine (250 mL daily) to patients receiving water. Physical, clinical, and blood parameters were evaluated on Days 1 and 14. The data show a positive effect of low wine consumption on blood parameters (decrease in total cholesterol and LDL; increase in erythrocyte membrane fluidity and antioxidant status). The results show that a moderate consumption of red wine even for a short period associated with a "Western prudent" diet improves various blood parameters in lipid and anti-oxidative status in patients with previous coronary ischemic accidents.

Incidence of Abcd1 level on the induction of cell death and organelle dysfunctions triggered by very long chain fatty acids and TNF-α on oligodendrocytes and astrocytes.

X-linked adrenoleukodystrophy (X-ALD) is characterized by ABCD1 deficiency. This disease is associated with elevated concentrations of very long chain fatty acids (C24:0 and C26:0) in the plasma and tissues of patients. Under its severe form, brain demyelination and inflammation are observed. Therefore, we determined the effects of C24:0 and C26:0 on glial cells:oligodendrocytes, which synthesize myelin, and astrocytes, which participate in immune response. So, 158N murine oligodendrocytes, rat C6 glioma cells, rat primary cultures of neuronal-glial cells, and of oligodendrocytes were treated for various periods of time in the absence or presence of C24:0 and C26:0 used at plasmatic concentrations found in X-ALD patients (1-5 µM) and higher (10, 20, 40 µM). To evaluate the importance of extrinsic and intrinsic factors, the part taken by TNF-α and reduced Abcd1 level was studied. Whatever the cells considered, no effects on cell growth and/or viability were detected at 1-5 µM, more or less pronounced effects were identified at 10 µM, and an induction of cell death with increased permeability to propidium iodide and loss of transmembrane mitochondrial potential was observed at 20-40 µM. On 158N, cell death was characterized by (i) an increased superoxide anion production at the mitochondrial level; (ii) the presence of vacuoles of different sizes and shapes; a destabilization of lysosomal membrane and a cytoplasmic redistribution of lysosomes; (iii) a modulation of Abcd3/PMP70 and Acox-1 protein expression, and a decrease in catalase activity at the peroxisomal level. When TNF-α was combined with C24:0 or C26:0 and used on 158N cells, C6 cells, and on 158N cells after siRNA mediated knockdown of Abcd1, no or slight potentiation was revealed. Thus, on the different cell models used, an induction of cell death with marked cellular dysfunctions at the mitochondrial, lysosomal, and peroxisomal levels were found with C24:0 and C26:0 at 20 µM and higher. However, in our experimental conditions, plasmatic concentrations of these fatty acids were unable to induce cell death, and organelle dysfunctions on oligodendrocytes and astrocytes, and additional intrinsic and environmental factors, such as reduced Abcd1 level and/or TNF-α, were ineffective to potentiate their side effects.

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes.

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 µg/mL corresponding to 50 µM, 24-48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3ß), and with degradation of Mcl-1. With α-tocopherol (400 µM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3ß dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3ß/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.

Analysis of the capture and influence of nanoparticles on the cytotoxic effects of 7-ketocholesterol on cardiac cells: investigation by flow cytometry and by dynamic and spectral imaging microscopy associated with factor analysis of medical image sequences.

OBJECTIVE: To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine. RESULTS: Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles. CONCLUSION: The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.