An optimized method for intensive screening of molecules that stimulate beta-defensin 2 or 3 (hBD2 or hBD3) expression in cultured normal human keratinocytes.

Normal human skin controls the intrusion of microorganisms by the production of peptide antibiotics such as defensins. The aim of our study was to develop a culture model of normal human keratinocytes for optimal beta-defensin mRNA detection which allows the screening of molecules able to stimulate hBD2 and hBD3 without inducing pro-inflammatory cytokines. A keratinocyte culture model in 96-well plates, in high calcium medium (1.7 mm) allowed to analyze hBD2 and hBD3 mRNA expression in basal condition and after cell stimulation by products from diverse vegetal extracts. The release of IL-8 and the chemokine MIP-3alpha was also evaluated in cell supernatants by ELISA. Among the 184 extracts tested, 75 showed a stimulatory effect on beta-defensin expression: 40 on hBD2, 26 on hBD3 and nine on both defensins. Fifteen of these substances which also induced the release of pro-inflammatory cytokines were eliminated. Among the other substances, four were selected and were analyzed in a dose-dependent study (n = 4) by real-time quantitative RT-PCR and completed by a measure of MIP-3alpha, IL-8 and IL-1alpha levels. These data underline the important necessity of screening result controls by a quantitative method reproduced at least three times. This new method of intensive screening allowed us to exhibit vegetal extracts that were able to stimulate epidermal beta-defensin expression without inducing an up-secretion of pro-inflammatory cytokines.

Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells.

In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-beta1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry. Results showed that a 2-day treatment with the representative allergens DNCB and NiSO(4) induced significant changes of most antigens while other chemicals such as balm of Peru (strong allergen), kathon (moderate allergen), cinnamic aldehyde (mild allergen) or the irritant SLS had no significant effect. In contrast, the 4-day treatment with allergens substantially improved the results. Indeed, despite a large variability according to the donors, the number of modified antigens was significantly higher with all the tested chemicals, except kathon, as compared to that observed with the irritant SLS. The present study indicates that, in this model, the screening of mild or moderate allergens requires both the consideration of many antigens and a prolonged time of incubation with the chemicals.

In vitro stimulation of synthesis of key DEJ constituents in a reconstructed skin model: a quantitative study.

Fighting skin ageing is one of the major targets of cosmetology research. However, traditional approaches to skin ageing using stimulation of basal keratinocyte proliferation and fibroblastic neosynthesis appear today to be incomplete, particularly considering changes occurring at the dermal-epidermal junction (DEJ) during the course of ageing. Unfortunately, the lack of in vitro model limits the exploration process of the phenomena of DEJ ageing, and particularly the evaluation of the changes of key components, that are laminin-5, types IV and VII collagens. The aim of this work was to provide an in vitro model of reconstructed skin, base for new dosage and identification methods for qualitative and quantitative analysis of the key components of DEJ. Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR were successfully applied to this model to analyse mRNA of laminin-5, types IV and VII collagens and their variation in 'young' and 'mature' reconstructed skin model. Finally, this model was used to test the activity of ingredients for cosmetic application, in order to modulate the expression of the major components of DEJ. To conclude, we demonstrated that this in vitro model of reconstructed young and mature skin provides a useful tool to get into the biology of the DEJ, key structure of the skin, and specifically into its dynamic changes during the ageing process.

Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.

The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.

Measurement of epidermal moisture content by magnetic resonance imaging: assessment of a hydration cream.

The moisture content of the epidermis was measured by magnetic resonance imaging (MRI), using transverse relaxation time. The spatial resolution was 86 microns, allowing a quantitative, accurate and localized determination of variations in epidermal hydration. The wrists of 15 volunteers were studied before and after application of a hydration cream. Results showed an increase of 15% of epidermal T2 after application of the cream. Moisture content curves varied according to different degrees of skin dryness. This study demonstrates that MRI is a useful tool in evaluation of epidermal hydration.

2D NMR studies and 3D structure of the parallel-stranded duplex oligonucleotide Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements. The NOE values were converted into distances by using the complete 190 x 190 relaxation matrix. The unnatural duplex Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) forms a parallel right-handed helix with Watson-Crick base pairing; the alpha and beta deoxyriboses adopt a 3'-exo conformation. The acridine moiety was found stacked up the C9-G9 base pair. The structure of the first seven base pairs of this duplex was found similar to that of the duplex alpha-d(TCTAAAC)-beta-d(AGATTTG), which we had already investigated [Lancelot, G., et al. (1989) Biochemistry 28, 7871-7878]. Since these structures were generated by using experimental NOE values obtained independently on macromolecules whose global correlation time was different (3.8 and 2.2 ns), we conclude that this comparison is a good test of the viability of our method to generate three-dimensional structures of oligonucleotides in solution. Starting from different initial conformations, we show that the NOE constraints allow one to reach the same final restrained conformation, taking into account implicitly the solvent effect.

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.

NMR studies of complex formation between the modified oligonucleotide d(T*TCTGT) covalently linked to an acridine derivative and its complementary sequence d(GCACAGAA).

The oligodeoxynucleotide d(TTCTGT) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phOsphate via a pentamethylene linker (m5). In order to avoid its hydrolysis by nucleases inside the cel., one of its phosphates (TpT) was substituTed with a neopentyl group. Complex formation between each of the two purified isomers and the complementary strand d(GCACAGAA) was investigated by nuclear magnetic resonance. The COSY and NOESY connectivities allowed us to assign all the proton resonances of the bases, the sugars (except the overlapping 5'-5'' resonances), the acridine, and the pentamethylene chain. Structural information derived from the relative intensity of COSY and NOESY maps revealed that the duplex d(T*TCTGT).d(GCACAGAA) adopts a B-type conformation and that the deoxyriboses preferentially adopt a 2'-endo conformation. The NOE connectivities observed between the protons of the bases or the sugars and the protons of the dye show the intercalation of the acridine between the base pairs. NOE connectivities as well as imino proton resonances show that, at room temperature, the C7 base and the G8 base belonging to two different duplexes are paired. The pseudoaxial and pseudoequatorial isomers were assigned, and the differences in stability of their complex with the complementary strand are discussed.

2D-NMR studies of the unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG).

The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG). NOE data provide evidence that both duplexes form stable right-helical duplexes with an anti-conformation on the glycosyl linkages and a Watson-Crick pairing. NOESY and COSY spectra allowed us to determine that alpha and beta deoxyriboses adopt a 3' -exo conformation.

Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex.

In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments. The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase. The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least. Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem.

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.