Clinical evaluation of anaesthetic-like effect of two dermocosmetic formulations containing Aquaphilus dolomiae extract-G3 in subjects with sensitive facial skin.

BACKGROUND: Sensitive skin is a common condition of hyper-reactivity to external stimuli, e.g. heat or abrasion. The symptoms are subjective but can be measured using validated emotional and technical methods. Avène water has several beneficial effects on the skin. In vitro studies indicated that the active component of this natural spring water, Aquaphilus dolomiae extract-G3 (ADE-G3), modulates cutaneous sensitivity via an anaesthetic-like mechanism. OBJECTIVES: To assess facial skin reactivity after repeated application of two formulations containing ADE-G3. METHODS: In open-label studies, healthy subjects with sensitive facial skin applied cream or balm twice daily for 84 days. The severity of skin sensitivity was measured using the Sensitive Scale (based on quantifying visible or subjective signs). Subjective responses associated with pain or uncomfortable feeling were assessed by measuring electrodermal response (EDR). This involves measuring variations in skin electrical resistance due to non-conscious physiological changes in activity of the sympathetic nervous system. Subjects were also evaluated for beneficial effects according to a quantitative approach using semantic assessment of a question regarding their skin quality. Evaluations were performed before and after the first application, and after 29/30, 56 and 84 days of twice daily use. RESULTS: There was a significant decrease in the EDR after stimuli immediately after the application of both ADE-G3 formulations, which continued to decrease over 84 days (40-50% decrease by D85). Likewise, all physical and functional signs of the Sensitive Scale were significantly decreased immediately after the first application and at all time points tested after treatment. Verbatim analysis revealed a semantic shift, from mainly negative terms on D1 to mainly positive terms at D85 for both tested products. CONCLUSIONS: These results demonstrated that two formulations containing ADE-G3 reduced skin sensitivity, indicating a decreased activation of the sympathetic nervous system associated with this condition.

Protective properties of Avène Thermal Spring Water on biomechanical, ultrastructural and clinical parameters of human skin.

BACKGROUND: Thermal Spring Water (TSW) has been recognized to have beneficial effects on skin; however, the mechanisms underlying these are not completely elucidated. AIMS: We compared the effects of Avène TSW with mineral-rich (MR) TSW on the biomechanical properties of the skin using mechanistic ex vivo assays and clinical studies. METHODS: Ex vivo studies included the effect of both TSWs on the structure of the surface of human skin explants using scanning electron microscopy (SEM); mineral elemental content on the skin surface using SEM coupled to energy dispersing X-ray spectroscopy; and the stress properties of the stratum corneum (SC) when exposed to dehydration. Human clinical studies were conducted to compare the soothing effect of TSWs after a dermatological chemical peeling of face skin and to evaluate the overall sensitive scale of consumers using Avène TSW for 7 days. RESULTS: Both TSWs preserved surface skin ultrastructure; however, crystals formed from MR-TSW were needle-like and formed small grains, present in clusters heterogeneously spread over the surface. Needle crystals were mainly composed of calcium, while small clusters were mainly composed of sulphur. By contrast, Avène TSW-formed crystals composed of sodium and chlorine only were regular in shape and homogeneously distributed across the skin surface. Peak stress of SC layers was increased by MR-TSW, whereas Avène TSW showed a comparatively reduced effect on dehydration and stress. The difference in the two TSW types was reflected in clinical findings comparing postpeeling redness after TSW application. Avène TSW significantly decreased postpeeling redness, while MR-TSW increased it. The overall sensitive scale of consumers was decreased by 47% using Avène TSW for 7 days. CONCLUSIONS: Avène TSW decreases postpeeling redness and soothes sensitive skin in human volunteers. Mechanistic studies suggested that differences in biomechanical effects could be linked to differences in calcium content of the TSW.

On-the-road driving performance after use of the antihistamines mequitazine and l-mequitazine, alone and with alcohol.

OBJECTIVE: Previous studies demonstrated that mequitazine produces mild sedation after single doses. Its enantiomer, l-mequitazine, has a stronger potency for the H1 receptor. The aim of the current study was to assess the effects of l-mequitazine and mequitazine, alone and with alcohol, on driving. METHODS: Twenty-five healthy volunteers were treated with l-mequitazine 2.5, 5.0 and 10 mg, mequitazine 10 mg and placebo, alone and in combination with alcohol in a double-blind crossover design. Driving performance was assessed using the standardized highway driving test in normal traffic. Its primary measure is the Standard Deviation of the Lateral Position (SDLP). Secondary measures consisted of an auditory word learning test during driving, and subjective measures of driving performance. RESULTS: L-mequitazine 2.5 and 5.0 mg showed no effect on SDLP in the highway driving test, while SDLP significantly increased after l-mequitazine 10 mg (alone +1.59 cm; with alcohol +1.41 cm) and mequitazine 10 mg (with alcohol +1.17 cm). Alcohol significantly impaired all performance measures (SDLP +2.63 cm) but did not interact with the effects of treatment. Subjective measures indicated that participants were aware of the impairing effects of alcohol, but not of l-mequitazine and mequitazine. CONCLUSION: L-mequitazine can be considered safe to drive in dosages of 2.5 and 5.0 mg. L-mequitazine 10 mg led to mild driving impairment. Alcohol impaired all performance measures and added to the effects of l-mequitazine and mequitazine.

Involvement of interleukin-6 in atherosclerosis but not in the prevention of fatty streak formation by 17beta-estradiol in apolipoprotein E-deficient mice.

Interleukin-6 (IL-6) gene expressed in bone marrow-derived stromal cells and osteoblasts contributes to the state of mineralization and its control by estradiol may be involved in the development of post-menopausal osteoporosis. Since IL-6 is also expressed in the different cell populations of the arterial wall, the purpose of this study was to gain more insight into its involvement in the atherosclerotic process and the atheroprotective effect of estradiol by studying double deficient mice at the apolipoprotein E and IL-6 loci (IL-6(-/-)/E(-/-)). At 1 year of age, IL-6(-/-)/E(-/-) mice showed similar hypercholesterolemia to IL-6(+/+)/E(-/-) mice but presented significantly larger and more calcified lesions. In younger mice (sixteen weeks of age), no significant difference in fatty streaks could be detected in IL-6(+/+)/E(-/-), IL-6(+/-)/E(-/-) and IL-6(-/-)/E(-/-) mice on a normal chow diet. Estrogen supplementation at this age induced a decrease of fatty streak formation in all three genotypes. The combined data indicate that IL-6 expression is involved at the fibrous plaque stage of the atherosclerotic process but does not constitute a direct target for estradiol to prevent fatty streak formation.

Sequential substitution of 1,2-dichloro-ethene: a convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-.

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadecadienoic acids with high radiochemical and isomeric purities (>98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO(2) gave (9Z,11E)-[1-(14)C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (<2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.

Early fatal pontocerebellar hypoplasia in premature twin sisters.

We report clinical, neuroradiological and neuropathological findings of monozygotic twin sisters born at 30 weeks' gestation, with pontocerebellar hypoplasia (PCH) similar but not identical to type 2 PCH. They presented with hypertonia, jitteriness, spontaneous and provoked myoclonic jerks (hyperekplexia), apnoeic episodes, and progressive microcephaly. They died at 7 weeks of age from respiratory failure.

Large-scale preparation of (9Z,12E)-[1-(13)C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid and their [1-(13)C] all-cis isomers.

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z, 12E)-[1-(13)C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z, 15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-(13)C]-octadeca-9, 12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-(13)C]-octadeca-9,12, 15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised.

[Estrogens and the arterial wall]. / OEstrogènes et paroi artérielle.

Two isoforms of oestrogens receptor (alpha and B) have been identified in the cells of the arterial wall, and an heterogenity of their expression according to the animal species, to the vascular bed and to sex has been reported. Estrogens can thus directly influence the vascular physiology through a genomic mechanism, but extra-genomic mechanisms responsible for a short-term effect have also been suggested. Endothelium appears to be an important target for estradiol, because this hormone potentiates endothelium-dependant relaxation through an increase in NO bioavailability, and accelerates endothelial regrowth. In the model of apolipoprotein E-deficient mice, as the atrhroprotective effect deposit. The immune system appears to play a key role, as the athroprotective effect of estradiol is absent in mice deficient in T and B lymphocytes. Estrogens potentiate the endothelium-dependant relaxation through the increase in nitric oxide bioavailability. Endothelial dysfunction (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Estrogens prevent and even correct this endothelial dysfunction. In monkeys, this beneficial effect of estrogens is not altered by coadministration of progesterone, but is abolished.

Loss of atheroprotective effect of estradiol in immunodeficient mice.

Estradiol significantly decreases fatty streak formation in the aortic root of chow-fed apolipoprotein E-deficient mice. In contrast, immunodeficient mice with homozygous disruption at the recombinase activating gene 2 loci present fatty streak development that is insensitive to estradiol. Lymphocytes thus appear to be required for development of the atheroprotective effect of estradiol in this mouse model.

[Effect of estrogens on arterial physiology and mechanisms of their atherosclerosis-protective effect in animals]. / Effect des oestrogènes sur la physiologie artérielle et mécanismes de son effet athéroprotecteur chez l'animal.

Two isoforms of oestrogen receptors (alpha and beta) have been identified in the cells of the arterial wall, and a heterogeneity of their expression according to the animal species, the vascular beds and the sex has been reported. Oestrogens can thus directly influence the vascular physiology through a 'genomic' mechanisms, but 'extra-genomic' mechanisms responsible for a short-term effect have also been suggested. Oestrogens potentiate endothelium-dependent relaxation through an increase in nitric oxide bioavailability (increase in its production and/or decrease in its degradation by superoxide anion according to the vascular beds). Endothelial 'dysfunction' (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Oestrogen replacement prevents and even corrects this endothelial dysfunction. In monkeys, this beneficial effect of oestrogens is not altered by coadministration of progesterone, but is abolished by coadministration of medroxyprogesterone. Finally, oestrogens prevent fatty streak deposit, and the mechanisms of this atheroprotective effect are being studied.

[Effect of estrogens on arterial physiology and mechanisms of their atheroprotective action in the animal]. / Effet des oestrogènes sur la physiologie artérielle et mécanismes de leur effet athéroprotecteur chez l'animal.

The two isoforms (alpha and beta) of the oestrogen receptor were identified in arterial wall cells. The heterogeneity of their expression is considered according to vascular regions and gender. Oestrogens can have a direct influence on vascular physiology through a "genomic" mechanism of action, although "extragenomic" mechanisms allowing very short-term hormonal action are also possible. Oestrogens potentiate endothelium-dependent relaxation by increasing the bioavailability of nitrogen monoxide (higher production and/or lesser degradation by the superoxide anion as a function of vascular beds). The atheromatous artery is the site of endothelial "dysfunction" (an anomaly of endothelium-dependent vasodilation), which can be prevented and even corrected by administration of oestradiol. In the monkey, this beneficial effect of oestrogens is not altered by addition of progesterone, but abolished by the addition of medroxyprogesterone. Finally, oestrogens prevent formation of the fatty streak. Studies of the mechanism(s) of this effect are now in progress.

Expression of the interleukin-6 gene is constitutive and not regulated by estrogen in rat vascular smooth muscle cells in culture.

Vascular smooth muscle cells (SMC) are major constituents of the medial layer of blood vessels and are involved in the development of atherosclerotic plaque. SMC secrete copious IL-6 under basal conditions that can be increased by cytokines such as tumor necrosis factor-alpha and interleukin-1beta (IL-1beta). The goal of our studies was to define the role of estrogen in IL-6 production by SMC. In a first series of experiments, the expression of specific messenger RNAs as well as the production of IL-6 bioactivity by rat SMC in culture could be demonstrated in basal and IL-1-stimulated conditions, but was unaffected by estrogen treatment. Different constructs containing deleted or mutated fragments of the human IL-6 promoter driving luciferase or chloramphenicol acetyltransferase reporter gene were then transiently transfected in these cells. A significant basal activity that was increased 2- to 4-fold after IL-1beta stimulation was observed with the total IL-6 promoter. Deletion analysis indicated that the -158/+11 region containing activator protein-1 and cAMP response element sites was apparently the minimal region of IL-6 promoter to confer both constitutive and IL-1-inducible activities. Site-directed mutagenesis experiments suggest that basal activity is dependent upon the promoter sequence -158 to -112 containing the nuclear factor (NF)-IL6(-153) and Sp1 sites, whereas IL-1beta stimulation would depend on the residual -112 nucleotides containing NF-IL6(-75) and NF-kappaB sites. In contrast to the down-regulation of IL-6 expression by estrogen described in osteoblasts, ethinyl estradiol as well as 17beta-estradiol did not influence stimulated IL-6 activity in our experimental conditions whatever the construct tested, even when either estrogen receptor alpha or beta was overexpressed. Thus, the atheroprotective properties of estrogen are probably not mediated through the regulation of IL-6 production by SMC.

Inhibition of interleukin-6 promoter activity by the 24 kDa isoform of fibroblast growth factor-2 in HeLa cells.

The expression of the interleukin (IL-6) gene can be regulated by various activating or inhibitory stimuli. This modulation involves several regulatory binding sites on the IL-6 promoter, and appears to be in general cell-specific. We have previously described that the nuclear 24 kDa isoform of fibroblast growth factor-2 (FGF-2) is able to increase IL-6 gene expression in NIH-3T3 cells. The transduction pathway involved was shown to be distinct from the extracellular mode of action of the smallest 18 kDa FGF-2 isoform. In the present study, we show that 24 kDa FGF-2-encoding vectors transfected into HeLa cells inhibit various co-transfected constructs incorporating the promoter element of the IL-6 gene and either the luciferase or the chloramphenicol acetyltransferase units. This down-regulation occurs dose-dependently with the 24 kDa FGF-2, is IL-6-promoter-specific, and does not involve an autocrine loop of the growth factor, since exogenously added FGF-2 fails to modulate the IL-6 promoter activity. Furthermore, 24 kDa FGF-2 inhibits the activity of both the co-transfected deletion mutants IL-6(-224) and IL-6(-158), and the point-mutated IL-6 promoter constructs in which the activating protein-1, nuclear factor (NF)-IL-6 and NF-kappaB elements are disrupted. We identify a responsive region to 24 kDa FGF-2 between positions -158 and -109 on the IL-6 promoter, which notably contains a retinoblastoma control element.

Cell transformation results in the loss of the density-dependent translational regulation of the expression of fibroblast growth factor 2 isoforms.

Alternative initiation of translation at three CUG and one AUG start codons leads to the synthesis of four isoforms of fibroblast growth factor 2 (FGF-2) that have distinct intracellular localizations and affect the cell phenotype differently. We show here that the expression of FGF-2 CUG-initiated isoforms decreases in a cell-density-dependent manner in normal human skin fibroblasts (HSFs) concomitantly with the FGF-2 mRNA level. In contrast, CUG-initiated FGF-2 expression is constitutive in SK-HEP-1 cells and in HSFs transformed with SV40 large T antigen. Cell transfection using a plasmid containing the FGF-2 mRNA leader fused to chloramphenicol acetyl transferase demonstrated that up-regulation of the CUG codons depends on cis-elements located in this leader. Furthermore, UV cross-linking experiments revealed a correlation between CUG codons utilization and the binding of several proteins to the mRNA leader. On the basis of the presence of an internal ribosome entry site (IRES) in the FGF-2 mRNA, we used bicistronic vectors to transfect normal and transformed cells. The density-dependent regulation in normal HSFs was cap-dependent, whereas the constitutive CUG-initiated FGF-2 expression in transformed cells occurred essentially by an IRES-dependent mechanism. Unexpectedly, the use of the AUG start codon occurred exclusively by internal entry, which suggests the presence of a second independent IRES in the FGF-2 mRNA that would be constitutive. A study of the eIF-4E levels and of the 4E-BP1 phosphorylation state at increasing cell densities showed a decrease of the eIF-4E level, concomitant with 4E-BP1 dephosphorylation in normal cells but not in transformed cells. These data point out a complex mechanism for the regulation of FGF-2 isoforms expression involving both the cap-dependent and the cap-independent initiation of translation and favor a positive role of CUG-initiated FGF-2 in cellular proliferation and transformation.

Differential effects of interleukin-1 receptor antagonist and tumor necrosis factor binding protein on fatty-streak formation in apolipoprotein E-deficient mice.

BACKGROUND: The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. METHODS AND RESULTS: Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17beta was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. CONCLUSIONS: IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility.

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells.

OBJECTIVE AND METHODS: Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO* production from endothelium has been extensively characterized, little is known about endothelium-derived O2-*. In the present study, we determined the O2-* production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy. RESULTS: An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.

Endogenous basic fibroblast growth factor isoforms involved in different intracellular protein complexes.

Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins.

Radioresistance induced by the high molecular forms of the basic fibroblast growth factor is associated with an increased G2 delay and a hyperphosphorylation of p34CDC2 in HeLa cells.

The basic fibroblast growth factor-(bFGF) mediated signal transduction pathway has been implicated in cellular resistance to ionizing radiation. bFGF is synthesized from the same mRNA in four isoforms resulting from alternative initiations of translation at three CUG start codons (24, 21.5, and 21 kDa) and one AUG start codon (18 kDa). We analyzed the implication of high- and low-molecular forms of bFGF in radioresistance acquisition. For this, we transfected HeLa cells with retroviral vector containing either the CUG-initiated 24-kDa molecular form (HeLa 3A cells), the AUG-initiated 18-kDa molecular bFGF form (HeLa 5A cells), or the vector alone (HeLa PINA cells). A significantly increased radioresistance was obtained only in HeLa 3A cells (Dq = 810 +/- 24 cGy) compared with wild-type cells (Dq = 253 +/- 49 cGy) or HeLa PINA cells (Dq = 256 +/- 29 cGy; P < 0.001). This radioprotective effect was independent of an inhibition of radiation-induced apoptosis but related to an increased G2 duration after irradiation and to an hyperphosphorylation of p34cdc2 kinase. Knowledge of the high-molecular bFGF form-induced radioresistance pathway could offer novel targets for decreasing the radioresistance phenotype of tumors expressing high amounts of bFGF, such as glioblastoma.