Compounds, compositions and corresponding uses for preventing and/or treating of dyslipidemia

Abstract

The present invention relates to the field of medicine. It relates more particularly to the use of compounds to prevent and/or treat lipotoxicity in a subject, especially lipotoxicity by hypoxia. The invention relates more particularly to compositions, especially pharmaceuticals compositions and nutritional supplements or complements comprising such compounds as well as their use to prevent and/or treat lipotoxicity, especially lipotoxicity by hypoxia. The compounds and compositions of the invention can especially be advantageously used to prevent and/or treat a pathology from among pulmonary pathologies, especially cystic fibrosis or a chronic obstructive pulmonary disease.

Claims

1. A method for treating a subject with cystic fibrosis or chronic obstructive pulmonary disease, comprising the step of administering to said subject a composition comprising mannide monooleate.

2. The method according to claim 1, characterized in that the compound administered to the subject is non-toxic for cells capable of synthesizing neutral lipids.

3. The method according to claim 1, where the subject is an animal.

4. The method according to claim 1, wherein the compound is formulated as a composition being chosen from among a pharmaceutical composition and a nutraceutical or food supplement.

5. The method according to claim 1, wherein the composition further comprises N,N-diethanololeamide.

6. The method according to claim 1, wherein the composition further comprises 3-hydroxy-2,2-bis (hydromethyl)propyl oleate.

7. The method according to claim 2, characterized in that the compound administered to the subject is non-toxic for cells capable of synthesizing triglycerides and/or esterified sterols.

8. The method according to claim 7, characterized in that the compound administered to the subject is non-toxic for bronchial epithelial cells.

9. The method according to claim 1, wherein the subject has cystic fibrosis.

10. The method according to claim 1, where the subject is a mammal.

11. The method according to claim 1, where the subject is a human being.

12. The method according to claim 1, wherein the subject has chronic obstructive pulmonary disease.

Description

DESCRIPTION OF THE FIGURES

(1) The following figures are described:

(2) FIG. 1: Secretory pathway and membrane plasticity

(3) Following their synthesis, the membrane proteins or secreted proteins (molecular “tools” of the cells) must undergo steps of maturing within the cells. Each of the steps of this process known as the “secretion process” takes place in a specific sub-cell compartment (endoplasmic reticulum (ER) and Golgi apparatus especially). Obtaining mature proteins therefore requires functional intra-cell transport between the different endomembrane systems. This stream is influenced inter alia by the plasticity of the membranes of the intra-cell compartments which is itself directly correlated to the nature of the phospholipids (PL) which form the membranes. In particular, it is accepted that the presence of SFA in PL reduces the membrane fluidity whereas the PLs sheltering UFAs form more fluid membranes.

(4) The beneficial effect of oleic acid (Ole) is observed on the cells having the capacity to buffer an excess exogenous UFAs in the form of neutral liquids (triglycerides (TG) or esterified sterols (SE) stored in a form of lipidic droplets (GL)). In the cells not having this capacity, the surplus exogenous oleic acid ultimately leads to a proliferation of intra-cell membranes in prompting cell stress which will trigger apoptosis.

(5) FIG. 2: The UPR pathways in the higher eukaryotes (Pineau & Ferreira, 2010)

(6) FIG. 3: Oleic acid, OAG and LPA restore the growth of lip o-intoxicated yeasts

(7) FIG. 3A—Structure of oleic acid, OAG and LPA.

(8) FIG. 3B—The hem1Δ yeasts were cultivated in conditions of SFA accumulation as indicated. 5 μl drops of OAG, LPA and oleic acid in the concentrations indicated were then deposited on the surface of the agar medium. The restoration of growth of the hem1Δ yeasts is observed in the formation of colonies after three days.

(9) FIG. 4: OAG and LPA are not toxic for cells that do not synthesize triglycerides

(10) 5 μl drops of OAG, LPA or oleic acid were deposited, from stock solutions in the concentrations indicated, on the surface of an agar medium on which the QM strain had been preliminarily spread. After three days, haloes of growth inhibition (absence of colonies) can be observed in the case of oleic acid. These haloes are on the contrary not observed in the presence of LPA and OAG.

(11) FIG. 5: OAG and LPA reduce the UPR response in lipo-intoxicated yeasts

(12) A plasmidic construction carrying a fusion gene, corresponding to the encoding sequence of the gene LacZ placed under dependence of an artificial promoter containing four UPR elements (UPRE) was introduced into a hem1Δf yeast strain, as described in Pineau et al. (2009). During an induction of the UPR response, the Hac1p/XBP1p transcription is activated and gets fixed on the UPR elements of the fusion gene, leading to the transcription of the LacZ gene. With LacZ encoding β-galactosidase, the level of induction of UPR is measured to detect the corresponding enzyme activity. The hem1Δ yeast strain was cultivated in a liquid medium inducing the accumulation of SFA without other addition (Ø), or in the same medium supplemented with 200 μM of oleic acid, OAG or LPA as indicated. A.U.: arbitrary units.

(13) FIG. 6: OAG does not restore the production of di-unsaturated phospholipids, unlike oleic acid, and LPA, in lipo-intoxicated yeasts.

(14) The hem1Δ yeasts were cultivated in standard conditions (control) or in conditions of SFA accumulation without (Ø) or with the addition of 100 μM of oleic acid, LPA or OAG as indicated.

(15) FIG. 6A—After 7 h of incubation, the phospholipids (PL) were extracted and the different species of phosphatidylcholine (PC), which constitute the predominantly present PL were analyzed by mass spectrometry in positive mode, according to Pineau et al. (2008). The species 36:2 corresponding to a PC containing two oleic acid chains was regulated As can be seen, the addition of oleic acid or LPA results in an increase of the level of this species, which is not the case for OAG.

(16) FIG. 6B—All the species of PL detectable in these conditions of mass spectrometry were analyzed to globally quantify the content in SFA forms. The index of saturation thus obtained showed that OAG, unlike oleic acid and LPA, does not restore an index of saturation comparable to the one observed in the control condition.

(17) FIG. 7: OAG and LPA prevent apoptosis of the pancreatic β-cells in the presence of saturated fatty acids, in reducing the induction rate of UPR.

(18) The BRIN-BD11 pancreatic β-cells were cultivated in controlled conditions or in the presence of an exogenous source of saturated fatty acids (palmitic acid 200 μM), as described by Dhayal& Morgan (2011) in order to generate conditions of lipotoxicity with or without addition of OAG or LPA.

(19) FIG. 7A—The proportion of dead cells was estimated in the absence (Ctrl) or in the presence of palmitic acid (Palm), for growing concentrations of OAG and LPA.

(20) FIG. 7B—The rates of phosphorylation of eIF2α were also analyzed under different conditions by Western blotting method, in the absence (Ø) or in the presence of OAG or LPA and normalized with quantities of total eIF2α. Since the phosphorylation rate is correlated with the intensity of the UPR response, this experiment shows that OAG reduces the UPR induced by the accumulation of palmitate. A.U.: arbitrary units.

(21) FIG. 8: The bronchial tissues of patients affected by cystic fibrosis or COPD are lipo-intoxicated by SFA.

(22) FIG. 8A—Pulmonary biopsies were dissociated in order to keep only the bronchial component (left to right sequence).

(23) FIG. 8B—The total lipids of the bronchial epithelial cells were extracted, the phospholipids were purified and then the different species of phosphatidylcholine (PC), that constitute the predominantly present PLs were analyzed by mass spectrometry in positive mode according to Pineau et al. (2008). The species 32:0 and 36:2, correspond to PCs containing two palmitic acid chains and two oleic acid chains. As can be seen, the SFA/UFA ratio gets inverted when the PCs from controlled biopsies are compared with PCs from biopsies of patient/s affected by cystic fibrosis or COPD.

(24) FIG. 9: Induction of lipotoxicity by SFA in bronchial epithelial cell lines, 16HBE and CFBE

(25) FIG. 9A—The human bronchial epithelial cell lines, 16HBE and CFBE, were used to test the fatty acid content of their phospholipids in conditions of in vitro culture. Unlike the lipidomic profile observed for pulmonary biopsies of patients with cystic fibrosis or COPD (see FIG. 7B), the SFA/UFA ratio in the PC of the 16HBE and CFBE lines indicate an absence of lipotoxicity.

(26) FIG. 9B—CFBE cells were cultivated for 16 h with increasing quantities of palmitic acid. The analysis of the lipidomic profile of the cells in such conditions reveal that exogenous inputs in SFA mimic the lipotoxicity observed among patients affected by cystic fibrosis (100 μM Palm) or COPD (250 μM Palm).

(27) FIG. 10: Conditions of hypoxia induce in vitro the lipotoxicity of the 16HBE and CFBE cell lines artificially reconstituting the hypoxic lipotoxicity observed in the biopsies of patients

(28) 16HBE and CFBE were cultivated for 24 h under standard conditions (normoxia: 95% O.sub.2+5% CO.sub.2) or in an anoxic environment (hypoxia: 95% N.sub.2+5% CO.sub.2). After extraction of the total lipids and purification of the phospholipids, the overall fatty acid content was analyzed. The SFA/UFA ratios were calculated in order to determine the rates of saturation of phospholipids in each of these conditions. The results indicate that lipotoxicity can be induced in vitro by artificial hypoxia.

(29) FIG. 11: The anti-SFA compounds against the pro-apoptopic influence of lipotoxicity in bronchial epithelial cells

(30) CFBE was cultivated under standard conditions (control) or subjected to a source of exogenous SFA (palmitic acid 250 μM), for 16 h without (Ø) or with the addition of 100 μM of the compounds of interest as indicated. The cells were then lysed and the apoptosis was quantified as indicated in the part entitled “Examples”.

(31) FIG. 12: Mannide monooleate dissipates the bronchial hypertension of the pathological tissues

(32) FIG. 12A—Bronchial rings of healthy patients (control), patients affected by cystic fibrosis or chronic obstructive pulmonary disease (COPD) were dissected and then their basic tone was measured. The results show that the two respiratory pathologies are correlated with hypertension of the bronchial tubes.

(33) FIG. 12B—The same operation was carried following a pre-incubation of the rings for 4 h at 37° C. in a physiological solution supplemented (+) or not supplemented with 100 μM of mannide monooleate, as indicated.

(34) For each histogram, a ratio N/n is displayed. N corresponds to the number of rings tested and n to the number of patients analyzed.

EXAMPLES

(35) The invention will be understood more clearly from the following examples:

(36) A/ Yeast Strains, Mammal Cell Lines and Biological Samples

(37) The Saccharomyces cerevisiae yeast strains listed in Table 1 are used for the different tests of growth restoration, in order to reveal toxicity, for the analysis of the fatty acid content of the cell phospholipids as well as for the tests for triggering the unfolded protein response (UPR).

(38) The state of activation of UPR and the induction of cell death by lipotoxicity was also analyzed in pancreatic β-cell lines of rats, BRIN-BD11.

(39) Besides, the induction of apoptosis, the secretion of IL-8 and the lipidomic profile in response to lipotoxicity under SFA were also analyzed on human bronchial epithelial cell lines 16HBE (wild homozygote for the CFTR gene) and/or CFBE (homozygote F508del-CFTR).

(40) In addition, ex vivo experiments were performed on biopsies of healthy patients and patients affected by cystic fibrosis or COPD in order to determine the corresponding lipidic profiles as well as the influence of the compounds of interest on the phenomenon of pulmonary hypertension. The uses of biopsies comply with an ethical chart defined by the French equivalent of the Research Ethical Committees called Comité de Protection des Personnes Ouest III or CPP Ouest III.

(41) TABLE-US-00001 TABLE 1 Yeast strains used Strains Genotype Origin hem1Δ MATa trp1 his3 ura3 leu2 hem1::LEU2 FY1679α × FYHO4 QM (H1246 MATαare1::HIS3 are2::LEU2 ScanBi Ltd., W303) dga1::KanMX4 lro1::TRP1 ADE2 ura3 Alnarp, Sweden WT (G175 MATa ADE2 MET his3 leu2 ura3 trp1 ScanBi Ltd., W303) Alnarp, Sweden

(42) B/ Lipotoxicity of Hem1Δ Yeasts

(43) The strain carrying the hem1Δ strain is cultivated, under aerobic conditions, under stirring and at 28° C., in a YPG.sup.A liquid medium (YPG (yeast extract 1% (m/v), peptone 1% (m/v) and glucose 2% (m/v)) supplemented with δ-aminolevulinic acid (ALA) at 80 μg/mL). Lipotoxicity by saturated fatty acids (SFA) is prompted by depletion of unsaturated fatty acids (UFA)—the synthesis of which is dependent on the presence of haem (prosthetic grouping of the enzyme Ole1p especially)—by transfer into YPG.sup.+ medium (YPG supplemented with ergosterol at 80 μg/mL to compensate for the depletion in sterol obtained under these conditions). The lipotoxicity can be induced in a solid medium YPG.sup.++agar 2% (m/v) in transferring 3500 cells (hem1Δ coming from a pre-culture in YPG.sup.A)/cm.sup.2 or *alternatively in a liquid medium in inoculating 2.Math.10.sup.6 cells/mL of YPG.sup.+. Classically, the effects of lipotoxicity on SFA are analyzed 7 h after the transfer into YPG.sup.+ medium. The capacity of a compound to counter the deleterious effects of lipotoxicity with SFA is for its part evaluated successively with the addition of this compound on (or in) the YPG.sup.+ transfer medium after seeding with cells.

(44) C/ Lipotoxicity of Pancreatic β-Cells of Rats by Palmitic Acid

(45) 1) Preparation of Lipidic Reagents:

(46) The lipidic species are prepared in ethanol and then complexed with bovine serum albumen (BSA initially devoid of fatty acids) by incubation for one hour at 37° C. The stock of palmitate is obtained by the addition of a volume of ethanol before the entire mixture is heated to 70° C. for homogenization. OAG and LPA solutions are prepared in ethanol 100% at ambient temperature. For incubation of mammal cells, the final concentrations of BSA and ethanol in the culture medium are respectively kept at 1% and 5% (m/v).

(47) 2) Tests of Cell Viability:

(48) The pancreatic β-cell cell line (BRIN-BD11) of rats is cultivated in an RPMI-1640 complete medium containing glucose at 11 mM and supplemented with 10% (v/v) of fetal calf serum (FCS), 2 mM of L-glutamine, 100 U/mL of penicillin and 100 μg/mL of streptomycin. For each experiment, the cells are initially seeded at a density of 0.5×10.sup.5 cells/mL in six-well dishes for 24 h. The full medium was then replaced by an equivalent devoid of FCS but containing a lipidic reagent of interest, in desired concentrations, complexed with BSA. In the case of the controlled conditions, identical quantities of BSA and ethanol were then used. At the end of the incubations, all the cells (dead and living) are collected and centrifuged at 300 g for 5 minutes. The cell wall was then put into suspension in 200 μL of medium and then the DNA of the dead cells (having lost the integrity of the plasma membrane) was marked with propidium iodide (PI) by adding 200 μL of a solution of PI at 20 μg/mL in FACS buffer (phosphate-buffered saline (PBS), 2% (v/v) FCS, sodium azide 10 mM). After incubation at 10 minutes on ice, the samples thus obtained are analyzed by cytometry in flux. A Beckman Coulter EPICS XL MCL is used for quantification, an FL3 channel serves to detect emissions from PI interposed in the DNA and the analysis is done by means of the software EXPO32 ADC (Applied Cytometry Systems, V 1.1 build 207).

(49) 3) Western Blotting:

(50) The cells BRIN-BD11 are seeded at a density of 0.5×10.sup.5 cells/mL in T25 flasks for 24 h. As indicated here above, the complete medium is then replaced by an equivalent devoid of FCS but containing the lipid reagents of interest. After 6 h of incubation, the extraction of the total proteins is done by means of a lysis buffer (Tris 20 mM, NaCl 150 mM, EDTA 1 mM and Triton-X 1% (v/v)) containing protease and phosphate inhibitors. These proteins are then subjected to electrophoresis in acrylamide gel 12% NuPAGE® Novex® Bis-Tris Gels (Invitrogen) and then transferred to a PVDF membrane and then probed by means of the antibody anti-phospho eIF2α (Cell Signalling (New England Biolabs)) diluted to 1/1000.sup.th. In a second stage, the membranes are stripped with the buffer Re-Blot Plus-Strong (Millipore) and then probed a second time with total anti-eIF2α antibodies (CellSignalling (New EnglandBiolabs) diluted to 1/1000.sup.th. The analysis by densitometry of the relative abundance of the phosphorylated and non-phosphorylated forms of the protein eIF2α is done with the Fluor-S Multi-imager analysis system combined with the software program Quantify One (Biorad UK Ltd).

(51) D/ Restoration of Growth

(52) 1) Screening of compounds: Following the induction of lipotoxicity with SFA (for hem1Δ cultivated in a solid medium), 5 μL drops of solutions of different compounds at 10 mM in dimethylsulfoxide (DMSO) or in ethanol (EtOH) are deposited on the surface of agar. The capacity of a compound to counter the lipo-induced stoppage of cell growth is estimated by the appearance of a halo of hem1Δ colonies at the position of the deposit of said compound after three days of culture at 28° C. (cf. Deguil et al., 2011).

(53) 2) Kinetics of proliferation: Conjointly with the induction of lipotoxicity with SFA (for hem1Δ yeasts in liquid medium), different compounds are added to the cultures at an initial concentration of 200 μM. The tracking of the proliferation is done by measuring the cell density by spectrometry at regular time intervals (every hour for the duration of the observation). At a wavelength of 600 nm, a unit of optical density (DO.sub.600 nm) corresponds to 2.Math.10.sup.7 cells/mL.

(54) E/ Toxicity Test

(55) At the same time, wild strains (WT) and QM are cultivated in aerobic conditions under stirring and at 28° C., in a YPG liquid medium before seeding 3500 cells per cm.sup.2 of YPG+agar 2% (m/v). Following this transfer to solid medium, 1 μL drops of solutions of different compounds at 1, 10 and 100 mM in DMSO or EtOH are deposited on the surface of the agar. Separately, deposits of DMSO and EtOH are also made in order to assess the intrinsic toxicity of these two solvents. After three days of culture of 28° C., the toxicity of the tested compounds is evaluated by comparing the diameters of the growth inhibition haloes obtained for the deposits of undissolved solvents with those of the deposits of different concentrations of compounds tested. Contrary to the WTstrain, the QM strain is incapable of buffering an excess of exogenous oleic acid in the form of neutral lipids (triglycerides (TG) or sterol esters (SE) in lipid droplets. Thus, in the case of an absence of toxicity relative to the WT strain, the observation of a toxicity of a compound relative to the QM strain indicates that this compound is perceived as a source of free fatty acid by the yeasts.

(56) F/ Extraction of Total Lipids

(57) The hem1Δ strain is cultivated in a YPG.sup.A, YPG.sup.+ or YPG.sup.+ liquid medium+200 μM of compound to be tested in aerobic conditions, under stirring and at 28° C. for 7 h, starting from an initial cell concentration of 2.Math.10.sup.6 cells/mL. At the end of the culture, 10.sup.8 cells are collected in order to carry out the extraction of the total lipids. After the cells have been put in suspension in 1 mL of distilled water at 4° C., 500 μL of glass beads (Ø 0.6 mm) are added and the whole mixture then undergoes three sequences of 20 seconds at 5000 rpm in a stirrer (the tubes are kept on ice between each of the three sequences). The cellular lysate then obtained, complemented with water for rinsing the beads (1 mL), is then transferred into a 40 mL glass tube (Corex™) and then the lipids are extracted by using a methanol:chloroform ratio of 2:1 (v/v). Initially, 6 mL of methanol are added and the entire mixture is vortexed for 30 seconds and then incubated for 15 minutes at 65° C. Once the mixture has been cooled to ambient temperature, 300 mL of chloroform is added and then the entire mixture is again vortexed for 30 seconds before allowing the extraction to take place for 16 h. Subsequently, the sample is centrifuged for 12 minutes at 10000 g and then the supernatant is transferred into a new Corex™ tube. After the addition of 2 mL of chloroform and then 4 mL of distilled water, the entire mixture is vortexed for 30 seconds and then centrifuged for 8 minutes at 3000 g. After elimination of the resultant upper phase, the lower organic phase is collected in a hemolysis tube made of glass. Finally, the solvent is evaporated under a nitrogen stream at 80° C. to obtain total cell lipid samples.

(58) In the case of biopsies on patients, after dissection of the pulmonary lobes and extraction of the bronchi, the bronchial epithelial cells were rinsed three times in PBS and then the total lipids were prepared as indicated here above, starting from 10.sup.5 cells.

(59) G/ Purification of Phospholipids and Analysis by Mass Spectrometry

(60) The samples of total cell lipids are put back into suspension in 1 mL of dichloromethane in being vortexed for 30 seconds. The entire mixture is deposited on a silica column (BOND ELUT-SI, 100 mg 1 mL) preconditioned with 3 mL of methanol and then 2 mL of dichloromethane successively. The fraction retained by the column is then washed with 2 mL of dichloromethane and then 2 mL of acetone successively. Finally, 2 mL of a mixture of chloroform/methanol/water 50:45:5 (v/v/v) are deposited on the column and the phospholipids thus eluted are collected in a glass hemolysis tube. The solvent is evaporated under nitrogen at 80° C. to obtain the samples of cellular phospholipids.

(61) Once put back into suspension in 100 μL of mixture Mix.sup.− (isopropanol/acetonitrile/water 2:1:1 (v/v/v)+triethylamine 1% (v/v)) or mixture Mix.sup.+ (isopropanol/acetonitrile/water 2:1:1 (v/v/v)+formic acid 1% (v/v)), the samples are analyzed by mass spectrometry (Electro Spray Ionization-Mass Spectrometry (ESI-MS)) in negative or positive mode respectively and the results obtained serve to analyze the fatty acid content of the different species of phospholipids.

(62) H/ UPR Triggering Test

(63) The hem1Δ strain transformed by the plasmid pPW344 [2 μ URA34×UPRE-LacZ (Patil et al., 2004)] is cultivated in liquid medium YPG.sup.A, YPG or YPG+200 μM of compound to be tested in aerobic conditions, under stirring and at 28° C. for 7 h, starting from an initial cell concentration of 2.Math.10.sup.6 cells/mL. At the end of the culture, 10.sup.8 cells are collected in order to quantify the beta-galactosidase (β-gal) activity resulting from the expression of the LacZ trans gene (in the case of a triggering of UPR). In a first stage, the cells are put back into suspension in 1.5 mL of buffer Z (Na.sub.2HPO.sub.4 at 60 mM, NaH.sub.2PO.sub.4 at 40 mM, KCl at 10 mM, MgSO.sub.4 at 1 mM and β-mercaptoethanol at 50 mM; solution at pH 7) then 1/15.sup.th of this suspension is used to carry out a measurement of DO.sub.600 nm. In a second stage, the suspension is complemented with 100 μL of sodium dodecyl sulphate (SDS) 0.1% (v/v) and 200 μL of chloroform then vortexed in two successive sequences of 30 seconds. After decantation (settling), 400 μL (volume V) of the solution thus obtained is transferred into a glass hemolysis tube and then complemented with 600 μL of buffer Z. 200 μL of ortho-nitrophenyl-β-galactoside (ONPG) substrate, at 4 mg/mL in the buffer Z is then added before the entire mixture becomes homogenized by vortex then incubated at 30° C. in a waterbath at 30° C. to initiate the reaction. When the entire mixture has a slightly yellowish color, the reaction is interrupted (at the time t) at ambient temperature by the addition of 500 μL of Na.sub.2CO.sub.3 at 1M. Finally, after the samples have been centrifuged for 5 minutes at 800 g and then the supernatants have been collected in the new glass hemolysis tubes, the products of the reaction (o-nitrophenol) as well as the cell debris are dosed by spectrometry at the wavelengths 420 and 550 nm respectively. For each sample, the activity β-gal (U) is computed using the formula U=(1000×[DO.sub.420 nm−(1.75×DO.sub.550nm)])/(t×DO.sub.600nm.), expressed in arbitrary units.

(64) I/ Lipotoxicity In Vitro of Bronchial Epithelial Cell Lines, 16HBE and CFBE

(65) 1) Lipotoxicity by exogenous palmitic acid. As with what has been presented here above for BRIN-BD 11, 16HBE and CFBE are cultivated in MEM supplemented by 5 μg/mL of plasmocine and 10% (v/v) of horse serum, at 37° C. and then exposed to palmitic acid concentrations of 50, 100 or 250 μM for 16 h. Then the consequences of the exogenous lipotoxicity are tested.

(66) 2) Hypoxic lipotoxicity. In standard way, the 16HBE and the CFBE are cultivated in conditions of normoxia. In this way, the cells are sustained in a chamber fed with a mixture formed by 95% of O.sub.2 and 5% of CO.sub.2. In the conditions of induction of hypoxia, the cells are subjected for 48 h to an anoxic gas mixture comprising 95% of N.sub.2 and 5% of CO.sub.2, and then the consequences of the lipotoxicity known as hypoxic lipotoxicity are tested.

(67) 3) Apoptosis test. Under conditions of exogenous lipotoxicity, the induction of apoptosis was analyzed by the use of the cell death detection kit ELISA.sup.PLUS (ROCHE). The CFBE are seeded in a 96-well dish at a concentration of 10000 cells per well. At the end of 16 h of lipotoxicity, the cells are lysed and the apoptosis is measured, according to the given instructions, by quantification of cytoplasmic oligonucleosomes which reveal DNA deterioration associated with apoptosis.

(68) J/ Measurement of Bronchial Basal Tone.

(69) After dissection of the pulmonary lobes, bronchial rings are isolated and mounted on a device for analysis by the technique of isolated organs, in which they are immersed in a KREBS physiological buffer. Following the stabilization of the tone of the circles, the basal tone is measured. As an alternative, the rings are incubated for 4 h in an additional KREBS buffer of 100 μM of mannide mono-oleate.

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