Broad spectrum repairing properties of an extract of Aquaphilus dolomiae on in vitro and ex vivo models of injured skin.

BACKGROUND: A biological concentrate was produced from cultures of an Avène aquatic microflora isolate, namely Aquaphilus dolomiae. Some of the beneficial effects on diseased and damaged skin are thought to be due to the presence of this microorganism. AIMS: An extract of A. dolomiae (A. dolomiae extract-G2, ADE-G2) was evaluated for its wound-healing effects using in vitro and ex vivo models of injured skin. METHODS: The effect of ADE-G2 on the proliferation of fibroblasts, migration of keratinocytes and re-epithelialization of ex vivo wounded skin explants was measured. Antimicrobial protection by ADE-G2 was measured by analysing the gene expression of a panel of antimicrobial proteins (AMPs) in keratinocytes (RNASE7, S100A7, DEFB4A/B and DEFb103B), as well as the protein encoded by DEFB4A-B (hBD2) in the medium. RESULTS: ADE-G2 increased fibroblast proliferation and keratinocyte migration, as well as re-epithelialization of wounded ex vivo skin. ADE-G2 induced the expression of all AMP genes analysed in keratinocytes, as well as stimulated the release in to the medium of hBD2 peptide, encoded by DEFB4A/B. CONCLUSIONS: We have shown the broad spectrum of the repairing properties of the A. dolomiae extract, ADE-G2. These results support the use of ADE-G2 as a promising component for use in formulations aimed at repairing skin, limiting wound superinfection and preventing complicated wounds.

Protective effect of Aquaphilus dolomiae extract-G1, ADE-G1, on tight junction barrier function in a Staphylococcus aureus-infected atopic dermatitis model.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. AIMS: Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. METHODS: An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). RESULTS: Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). CONCLUSIONS: ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Fragility of epidermis in newborns, children and adolescents.

Within their first days of life, newborns' skin undergoes various adaptation processes needed to accommodate the transition from the wet uterine environment to the dry atmosphere. The skin of newborns and infants is considered as a physiological fragile skin, a skin with lower resistance to aggressions. Fragile skin is divided into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. Extensive research of the past 10 years have proven evidence that at birth albeit showing a nearly perfect appearance, newborn skin is structurally and functionally immature compared to adult skin undergoing a physiological maturation process after birth at least throughout the first year of life. This article is an overview of all known data about fragility of epidermis in 'fragile populations': newborns, children and adolescents. It includes the recent pathological, pathophysiological and clinical data about fragility of epidermis in various dermatological diseases, such as atopic dermatitis, acne, rosacea, contact dermatitis, irritative dermatitis and focus on UV protection.

In vitro approaches to pharmacological screening in the field of atopic dermatitis.

In vitro models are valuable for evaluating potential active ingredients and other molecules used in medications for atopic dermatitis (AD). However, finding appropriate in vitro models can be problematic. Our strategy was to set up different in vitro models that would mimic the pathomechanisms of AD. We describe five such models - the AD keratinocyte model, the AD reconstructed human epidermis model, the adaptive immunity model, the innate immunity model and the pruritus model - which we have used to evaluate a new ingredient for emollients derived from a biological extract. The models chosen provide useful data for the pharmacological characterization of active ingredients in adjunctive treatments for AD.

Fragility of epidermis and its consequence in dermatology.

The skin is the largest organ of the body, providing a protective barrier against bacteria, chemicals and physical insults while maintaining homeostasis in the internal environment. Such a barrier function the skin ensures protection against excessive water loss. The skin's immune defence consists of several facets, including immediate, non-specific mechanisms (innate immunity) and delayed, stimulus-specific responses (adaptive immunity), which contribute to fending off a wide range of potentially invasive microorganisms. This article is an overview of all known data about 'fragile skin'. Fragile skin is defined as skin with lower resistance to aggressions. Fragile skin can be classified into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. This article includes the epidemiologic data, pathologic description of fragile skin with pathophysiological bases (mechanical and immunological role of skin barrier) and clinical description of fragile skin in atopic dermatitis, in acne, in rosacea, in psoriasis, in contact dermatitis and other dermatologic pathologies. This article includes also clinical cases and differential diagnosis of fragile skin (reactive skin) in face in adult population. In conclusion, fragile skin is very frequent worldwide and its prevalence varies between 25% and 52% in Caucasian, African and Asian population.

Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator.

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C(2)C(12) myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-alpha. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C(2)C(12) cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-beta/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C(2)C(12) cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.

Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion.

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.

Murine laminin alpha3A and alpha3B isoform chains are generated by usage of two promoters and alternative splicing.

We already identified two distinct laminin alpha3A and alpha3B chain isoforms which differ in their amino-terminal ends and display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of the same laminin alpha3 (lama3) gene and transcribed from one or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5' ends of both the alpha3A and the alpha3B cDNAs. Sequence analysis of the region upstream to the alpha3A open reading frame revealed the presence of a TATA box and potential binding sites for responsive elements. By primer extension analysis, the transcription start site of the alpha3B mRNA isoform was defined. The sequences upstream to the alpha3B mRNA transcription start site do not contain a TATA box near the transcription initiation sites, but AP-1, AP-2, and Sp1 consensus binding site sequences were identified. The genomic regions located immediately upstream of the alpha3A and alpha3B transcription start sites were shown to possess promoter activities in transfection experiments. In the promoter regions, response elements for the acute phase reactant signal and NF-interleukin 6 were found, and their possible relevance in the context of inflammation and wound healing is discussed. Our results demonstrate that the lama3 gene produces the two polypeptides by alternative splicing and contains two promoters, which regulate the production of the two isoforms alpha3A and alpha3B.

Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa.

Laminin 5 and laminin 6 are basement membrane proteins synthesized by the basal cells of stratifying squamous epithelia. Altered expression of laminin 5 has been associated with Herlitz junctional epidermolysis bullosa (H-JEB), a severe epidermal blistering disorder inherited as an autosomal recessive disease. We have isolated cDNA clones encoding the alpha 3 chain of laminin 5 and searched for mutations in the LAMA3 gene in H-JEB patients. In one H-JEB family, an affected individual exhibited drastically reduced immunoreactivity to antibodies directed against the alpha 3 chain of laminin 5 and an impaired expression of the corresponding mRNA transcripts. RT-PCR analysis of mRNA extracted from the proband's keratinocytes identified a homozygous single basepair deletion in the transcripts encoding the laminin alpha 3A and alpha 3B isoforms. The mutation causes a frameshift and premature termination codon in both alleles of the LAMA3 gene. Inheritance of the clinical H-JEB phenotype was consistent with the segregation of the mutated allele in the family. We also report the identity of the alpha chains of laminin 5 and epiligrin and provide evidence that LAMA3 transcripts are distinct from the laminin 6 alpha chain mRNA.

Cloning and complete primary structure of the mouse laminin alpha 3 chain. Distinct expression pattern of the laminin alpha 3A and alpha 3B chain isoforms.

We have isolated and characterized overlapping cDNA clones encoding the alpha 3A and alpha 3B chains of mouse laminin 5. Sequence analysis of the cDNA for the alpha 3B predicts a polypeptide of 2541 amino acids (279,510 Da) comprising a truncated short arm and a carboxyl-terminal long arm common to the laminin alpha chains identified thus far. The short arm of the alpha 3B chain harbors two alternating epidermal growth factor-like domains and two globular domains. The amino-terminal globular domain, thought to mediate interactions with molecules of the extracellular matrix, shows no significant homology to any globular domain at the tips of the known laminin isoforms. The alpha 3A cDNA predicts a polypeptide of 1711 amino acids (186,230 Da) that substitutes a short sequence of 43 amino acids for the short arm seen in the alpha 3B isoform and displays 77% conservative homology to the alpha 3Ep chains of the adhesion ligand epiligrin. Northern and Western blot analyses of skin and lung epithelial cells demonstrated the tissue-specific expression of the laminin alpha 3A and alpha 3B isoforms, and in situ hybridization on mouse embryos revealed a focal localization of alpha 3B in areas of the central nervous system.

Developmental expression of nicein adhesion protein (laminin-5) subunits suggests multiple morphogenic roles.

Nicein/kalinin (laminin-5) is a heterotrimeric laminin-like adhesion protein, which is secreted at the basement membrane of subsets of epithelia and is involved in the etiology of junctional epidermolysis bullosa, a severe human blistering disease characterized by disadhesion of epidermis from dermis. cDNA clones encoding the three chains of mouse nicein and antibodies specific to each polypeptide were used to examine the expression of the protein in the developing mouse embryo from 10.5 day post coitum to 7 days after birth. At various stages of development, co-expression of the three chains of nicein was observed in amnion, skin, and in epithelia of respiratory, urinary and digestive systems. High level expression of nicein was seen in enamel-secreting ameloblasts in developing teeth. Cell-specific distribution of nicein was also detected in other specialized tissues representative of the three primary embryonic germ layers with prominent secretory or protective functions. Differential and focal expression of nicein subunits was observed in the choroid plexus and the floor plate of the neural tube. Messenger for the heavy chain of nicein was detected in the floor plate, where mouse s-laminin messengers were also found. This suggests that nicein genes may play a role in the migration and polarization of motor neurons in the developing spinal cord.

Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).

We have linked Herlitz's junctional epidermolysis bullosa (H-JEB) to the gene (LAMC2) encoding the gamma 2 subunit of nicein/kalinin, an isolaminin (laminin-5) expressed by basal keratinocytes. In four H-JEB kindreds, a maximum two-point lod score of 5.33 at theta = 0 was observed between a microsatellite near LAMC2 at 1q25-31 and the disease. In one family, a homozygous point mutation leading to a premature stop codon (CGA to TGA) was identified in exon 3 of the gene. The segregation of the mutated allele implicates the mutation in the pathology of the disorder and corroborates the linkage results.