Inhibition of TNF-alpha induced-adhesion molecules by Avène Thermal Spring Water in human endothelial cells.

BACKGROUND: Cell adhesion molecules, such as E-selectin or intercellular adhesion molecule 1 (ICAM-1), play an important role in mediating leucocyte capture and rolling on the surface of blood vessels in atopic skin. The effectiveness of Avène hydrotherapy in patients suffering from atopic dermatitis has previously been demonstrated. Thus, we examined the effect of Avène Thermal Spring Water (TSW) on adhesion molecules to understand its mechanism of action. METHODS: Human endothelial cells EA.hy926 were treated with tumour necrosis factor-α (TNFα) in the presence or not of Avène TSW during 4 h. As nuclear factor-κB (NF-κB) is involved in the signalisation of inflammatory mediators such as the adhesion molecules, the translocation of NF-κB in endothelial cells was assessed by immunohistochemistry with anti-NF-κBp65. The protein and mRNA levels of TNFα-induced ICAM-1 and E-selectin were assessed by ELISA assay and RT-PCR. These adhesion molecules were also detected by immunohistochemistry. RESULTS: Tumour necrosis factor-α induced the activation of p65 NF-κB nuclear translocation. TNFα also induced E-selectin and ICAM-1 in a dose-dependant manner in EA.hy926 endothelial cells. In the presence of Avène TSW, a significant inhibition of the TNFα-induced E-selectin and ICAM-1 expression (-22% and -7%, respectively, P < 0.05) was observed. CONCLUSION: These data suggest that Avène TSW mediated inhibition of TNFα-induced E-selectin and ICAM-1 expression. The inhibition of such adhesion molecules is attributable to the suppression of NF-κB transcription factor pathway activation.

TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response.

Gingival tissue faces constant exposure to micro-organisms. It functions as part of the host response, an anti-microbial barrier that recognizes and discriminates between commensal and pathogenic bacteria. This study aimed to evaluate and compare the effects of cell wall extracts from different periodontal bacteria, commensals Streptococcus sanguinis and Fusobacterium nucleatum and the pathogen Porphyromonas gingivalis, on the innate immune response of gingival keratinocytes and the role of TLR2 in regulating this. We assayed mRNA levels to determine the expression of human beta-defensins (hbetaD2, hbetaD3), interleukin-1alpha, -1beta, 6 and 8 and matrix metalloproteinase-9. F. nucleatum extracts induced beta-defensin and inflammatory marker mRNA expression at higher levels than P. gingivalis. Extracts from the Gram-positive commensal S. sanguinis did not upregulate the host response. TLR2 extinction inhibited the upregulation of beta-defensin and cytokine transcripts by F. nucleatum extracts but, in contrast, led to a weak induction of hbetaD3 after challenge with S. sanguinis extracts. Although F. nucleatum strongly induces innate immune and inflammatory mediators, S. sanguinis limits their expression through TLR2. Together, our data demonstrate that gingival keratinocytes recognize and discriminate between Gram-positive and Gram-negative commensal extracts, in part through TLR2, to activate different signaling pathways of the innate immune host response.

Modulation of integrins and filaggrin expression by Propionibacterium acnes extracts on keratinocytes.

Propionibacterium acnes plays an important role in the pathogenesis of acne and it is established that this bacteria is involved in the induction and maintenance of the inflammatory phase of acne. The aim of our work was to determine if P. acnes extracts could modulate integrins and filaggrin in vitro expression by keratinocytes. Integrins and filaggrin expression was examined using immunohistochemistry technique both on Normal Human Epiderminal Keratinocytes (NHEK) and on deep-frozen sections of normal human skin explants incubated with three different P. acnes extracts. In addition, the expression of filaggrin was investigated on biopsies of acne lesions and by western-blot associated with its precursor profilaggrin. We demonstrated that P. acnes extracts induced beta1 integrin expression significantly on both proliferating keratinocytes and differentiated keratinocytes. In addition, P. acnes induced alpha3, alpha6s and alphaVbeta6 integrin expression and filaggrin expression on differentiated keratinocytes. Finally P. acnes extracts increased filaggrin expression by suprabasal layer of epidermis of explants. Western-blot confirmed that total amount of filaggrin was increased. These results indicate that P. acnes extracts are directly able to modulate the differentiation of keratinocytes suggesting that this bacteria play a role not only in the development of inflammatory acne lesions but also in the formation of the microcomedo.

Incorporation of phytosterols in human keratinocytes. Consequences on UVA-induced lipid peroxidation and calcium ionophore-induced prostaglandin release.

We have designed experimental conditions allowing the replacement of 50% of cholesterol of human keratinocytes (SVK14 line) with sitosterol or stigmasterol without affecting cellular viability. We have investigated the influence of incorporating phytosterol on the ultraviolet-A-induced formation of lipid-peroxidation products (thiobarbituric reactive substances (TBARS)) in these cells. Our results show that ultraviolet-A-induced lipid peroxidation depends on the nature of the phytosterol. Sitosterol induces a significant decrease (-30%) of TBARS relative to the control whereas stigmasterol markedly increases lipid peroxidation (+70%). We have also studied the effect of plant sterols on prostaglandin release by using the Ca(2+) ionophore A23187 as an in vitro model of the inflammation induced by UVA radiation. We show that in the presence of 50% of phytosterol (particularly stigmasterol), the release of prostaglandin (6-ketoPG(1alpha), PGE(2)) is increased compared to untreated cells. This pro-inflammatory effect of phytosterols is correlated with a loss of the regulation of the intracellular Ca(2+) concentration.

Identification of two secreted phospholipases A2 in human epidermis.

Phospholipases A2 are enzymes that catalyze the release of fatty acids from the sn-2 position of phospholipids. Fatty acids have been suggested to play a key role in the barrier function of the epidermis. The aim of this study was to identify and characterize the type of secretory phospholipase A2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A2. Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A2 is identical to human synovial type IIA enzyme and is localized in the membrane fraction. By semiquantitative reverse transcription-polymerase chain reaction performed on horizontal sections of the epidermis, we found that the mRNAs of both phospholipases A2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A2 in human epidermis. The different localization of these two secretory proteins strongly suggests that each enzyme might have a specific role in skin physiology and probably in the barrier function. Taken together, these data validate the reverse transcription-polymerase chain reaction technique performed on thin sections as a first approach to detect gene expression in different layers of the epidermis.

Vascular endothelial growth factor is an autocrine growth factor for hair dermal papilla cells.

The transition of the late anagen to the catagen phase is concomitant with the disappearance of perifollicular capillaries, and therefore cyclical hair growth might depend on the ability of the dermal papilla to synthesize and release soluble growth and differentiation factors toward pre-existing capillaries. We characterized an angiogenic growth factor in the conditioned medium of dermal papilla cells indistinguishable from vascular endothelial growth factor as judged by biochemical and immunologic criteria. In addition, these cells bind vascular endothelial, growth factor on two binding sites and proliferate or migrate in the presence of this growth factor. Moreover, neutralizing antibodies inhibit these biologic effects, confirming that vascular endothelial growth factor might contribute to hair growth either by acting directly on papilla cells or by stimulating the local vascularization.

Effects of prolonged administration of milnacipran, a new antidepressant, on receptors and monoamine uptake in the brain of the rat.

Most antidepressants produce changes in monoamine receptors in brain after chronic administration in animals. The most commonly described alterations are a decreased density and function of beta-adrenergic receptors and have been postulated to be the mechanisms by which antidepressants exert their therapeutic effect. Milnacipran (previous name midalcipran) is a new, clinically-effective antidepressant, which inhibits the uptake of both serotonin and noradrenaline but has no affinity for any pre- or postsynaptic receptor studied. When given either orally at 7.5 mg/kg twice daily for 3 days, at 30 mg/kg once daily for 3 weeks, by osmotic mini-pump at 30 mg/kg/day for 27 days, or in drinking water at approximately 15 mg/kg/day for 6 weeks and after a washout period of 24 hr, milnacipran produced no down-regulation of beta-adrenoceptors. In addition, there were no alterations of alpha 1- or alpha 2-adrenoceptors, 5-HT1, 5-HT2 receptors or benzodiazepine binding sites. Moreover, uptake and accumulation of serotonin and noradrenaline were unmodified. In addition, the potency for milnacipran to inhibit monoamine uptake in vitro in the cortex was not altered in treated rats, compared to control animals. Thus, in spite of its action on both the uptake of serotonin and noradrenaline, milnacipran appears to be without long-term action on beta-adrenoceptors or the other receptors studied, suggesting that, at least for this antidepressant, these modifications are not essential for clinical activity.

Biochemical profile of midalcipran (F 2207), 1-phenyl-1-diethyl-aminocarbonyl-2-aminomethyl-cyclopropane (Z) hydrochloride, a potential fourth generation antidepressant drug.

The present study of midalcipran (F 2207), 1-phenyl-1-diethyl-aminocarbonyl-2-aminomethyl-cyclopropane(Z) hydrochloride, was undertaken to determine its biochemical profile. The properties of midalcipran, in inhibiting the uptake of monoamines were tested and compared with that of imipramine. In vitro, midalcipran was found to inhibit the uptake of radiolabelled serotonin and noradrenaline (IC50 = 203 and 100 nM, respectively), but not that of dopamine, into brain slices. Hyperthermia induced by the centrally-acting displacers of monoamines, H77/77 and H75/12, were almost equipotently antagonized by midalcipran, confirming the inhibition of the uptake of serotonin and noradrenaline by midalcipran in vivo (ID50 = 11 and 4.8 mg/kg, respectively). Midalcipran showed no inhibition of the activity of monoamine oxidase in vitro or in vivo. The interaction between midalcipran and neurotransmitter receptors and binding sites in the CNS was studied in the rat in comparison with imipramine and desipramine. In contrast to these two antidepressant drugs, midalcipran showed no affinity for alpha- or beta-adrenoceptors, muscarinic, histaminergic H1, dopaminergic D2 or serotonergic 5-HT2 receptors, suggesting a general absence of anticholinergic, sedative and other side-effects. Midalcipran was equipotent with imipramine at inhibiting the binding of [3H]imipramine. Chronic administration of midalcipran to rats did not alter the number of beta-adrenergic receptors in the cortex, in contrast to imipramine and desipramine which decreased the binding of beta-adrenoceptors. Thus midalcipran appears to act exclusively presynaptically, inhibiting the uptake of serotonin and noradrenaline. This activity, coupled to the total absence of interaction at postsynaptic sites, suggests that midalcipran may be a useful and novel antidepressant drug.

In vivo [(3)H]ketanserin binding: effect of modifying synaptic serotonin levels.

Most antidepressant therapies aim to increase the synaptic concentration of one or more of the monoamines. Synaptic monoamine levels are, however, extremely difficult to measure. In order to estimate synaptic levels of serotonin, an indirect method has been used. Since [(3)Hjketanserin is an antagonist at the 5HT(2) serotonin receptor, one would expect its in vivo binding to be inhibited by increased levels of synaptic serotonin. This hypothesis was tested in mice by measuring the effect of compounds which are considered to raise the synaptic concentration of serotonin. Directly acting agents such as quipazine, methysergide and mianserin inhibited [(3)H]ketanserin binding in vitro and in vivo. On the other hand, indirect agonists such as the monoamine oxidase inhibitors, pargyline and niamide, the serotonin uptake blockers, paroxetine and midalcipran, the serotonin releasers, p-chloroamphetamine and H75/12 and the serotonin precursor, 5-hydroxytryptophan had no effect on [(3)H]ketanserin binding in vivo. This was in spite of the fact that at doses used a very marked serotonin-induced behaviour was observed. In view of its insensitivity to changes in synaptic concentrations of serotonin, it is possible that the sites labelled in vivo by [(3)H]ketanserin are not innervated by the serotonin nerve terminals through which these indirect serotonin agonists act.

Benzodiazepine agonist-type activity of raubasine, a rauwolfia serpentina alkaloid.

Raubasine produced a dose-related inhibition of specific [3H]flunitrazepam binding to rat brain membranes. Scatchard analyses revealed a significant increase in the affinity constant but no change in the number of binding sites, suggesting that raubasine acts as a competitive inhibitor. Raubasine also inhibited the in vivo binding of [3H]flunitrazepam to mouse brain sites. Behavioral studies showed raubasine to possess anticonvulsant properties against pentylenetetrazol- and bicuculline-induced convulsions in mice. These effects were inhibited by the benzodiazepine antagonist, Ro 15-1788. The results suggest that raubasine interacts directly at benzodiazepine sites with a benzodiazepine agonist-type activity.

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.

Effects of thermal shocks on interleukin-1 levels and heat shock protein 72 (HSP72) expression in normal human keratinocytes.

Interleukin-1 expression is reported to be modified under a number of cell conditions including physiological stress, injury and activation. We report the effects of the physiological stresses cold and heat shock on IL-1 levels in keratinocytes. Having observed that normal human skin obtained from plastic surgery, usually stored at 4 degrees C for a few hours, highly expressed HSP72, a constant feature of stressed human keratinocytes, we wondered whether this induction could be linked to a cold shock and to modification of IL-1 levels in keratinocytes. Cultured keratinocytes were incubated at 4, 37, 40 and 43 degrees C for 1.5, 4, 8 and 16 h in a defined medium. HSP72 expression was studied by immunohistochemistry and immunoblot and IL-1 was quantified using specific and sensitive radioimmunoassay. Our findings showed that intracellular IL-1 alpha and IL-1 beta levels are not significantly modified by thermal shock. HSP72 is only induced after cell exposure to 43 degrees C and is not a cold-shock protein. These results demonstrate that thermal stress is not an inductive signal for IL-1 modification in keratinocytes.

Effect of various metals on intercellular adhesion molecule-1 expression and tumour necrosis factor alpha production by normal human keratinocytes.

Nickel, cobalt and chromium are metals very often implicated in allergic contact dermatitis. In vivo, keratinocytes, which are the first target cells, can be directly activated to participate in the local reaction, especially through the expression of the membrane antigen ICAM-1, a ligand of the leucocyte antigen LFA-1, and the production of cytokines. Our aim was to assess the effects of sensitizing metal haptens (nickel, cobalt and chromium) compared with the toxic metal cadmium on the induction of ICAM-1 and the production of TNF alpha by epidermal cells. For this purpose, normal human keratinocytes obtained during plastic skin surgery were cultured in low-calcium defined medium (MCDB153) and the metals were used in non-toxic concentrations. Using FACS analysis, ICAM-1 expression was found to be induced only by nickel. This stimulation appeared as early as 24 h after stimulation. All the metals induced a low expression of TNF alpha detectable by immunocytochemistry correlating with the induction of the nuclear stress protein Hsp72 which is closely linked genetically with the TNF alpha locus. However, only Ni2+, Co2+ and Cr2+ induced a significant release of TNF alpha detectable by ELISA after 48 h stimulation. This secretion was lower than that observed with known stimulants such as lipopolysaccharide. These results indicate that the metals studied are able to induce an aggressive cellular effect, and that nickel, by its ICAM-1 induction, may play a major role in the keratinocyte activation state during allergic contact dermatitis.

Inhibitory effects of bFGF, VEGF and minoxidil on collagen synthesis by cultured hair dermal papilla cells.

Dermal papilla cells of rat vibrissa follicles cultivated in monolayers and in three-dimensional collagen gels show a different morphology in these culture systems. Dermal papilla cells cultured in lattices tend to express morphological features resembling those seen in vivo. Quantification of total collagen by incorporation of 3H-proline in monolayer cultures and in collagen lattices show that the amount of collagen found in dermal papilla cells is higher than that secreted. Moreover, collagen synthesis measured in lattices is reduced to about 50% of that found in monolayer cultures. The influence of growth factors on collagen synthesis by hair dermal papilla cells was investigated. We studied the effects of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and minoxidil on collagen synthesis in monolayers and in lattices. VEGF, bFGF and minoxidil significantly decreased the total amount of collagen. In monolayer cultures, there was approximately a 30% inhibition of collagen production with 5 ng/ml bFGF, 0.1 ng/ml VEGF and 100 ng/ml minoxidil. However, in the lattices this inhibition was reduced to about half. These results suggest that both culture substrate and growth factors influence collagen production by rat hair dermal papilla cells.

A case of dyskeratosis congenita associated with impaired DNA repair as shown by the comet assay.

An 18-year old boy with dyskeratosis congenita is presented. To examine the DNA metabolism of our patient, we applied the comet assay, a simple, quick and sensitive method that so far has not been used in this disease. After exposure to UVB, cells originating from the patient present abnormal DNA repair localized in the late step. We consider that such repair deficiency could be related to susceptibility to cancer. The comet assay seems to be a good procedure to investigate dyskeratosis congenita or other genodermatoses.

[Implication of VEGF, steroid hormones and neuropeptides in hair follicle cell responses]. / Rôles du VEGF, des hormones stéroïdes et des neuropeptides dans les réponses cellulaires des follicules pilaires.

Human hair follicles progress independently through the anagen, catagen, telogen and latency phases that correspond to growth arrest and hair shedding before initiation of a new anagen phase. Hair follicles are self-renewing and contain reservoirs of multi-potent stem cells. Identification of the messenger molecules and pathways operating in the growth and cycling of hair follicles, have provided substantial data. However, only a limited number of these signals is well understood. The specific response of hair follicle cells to these signals is correlated with the expression of their corresponding receptors. What regulates these responses? In this review, we will focus on the hair cycle and its control mechanisms. We will provide some elements in answer to these questions and present some of the markers of hair follicle cells, and hormonal and vascular growth factors, which may regulate respectively hair follicle cell metabolism and cycle, and the neuropeptide impact on hair follicle response and hair growth. The results of our study show the modifications in various expression patterns of receptors in dermal papilla cells, and demonstrate the cross-interaction between these different components. In conclusion, we present an accumulation of evidence suggesting that the regulation of hair growth requires a combination of hormonal, vascular and neuropeptide approaches that will provide further insight in defining new treatments for hair loss.