Owing to their high content of flavonoids and saponins, plantlets of Avena sativa L. (Poaceae) are likely to possess anti-inflammatory and immunoregulatory properties of value in the treatment of atopic dermatitis (AD). With a view to its potential use in atopic subjects at risk of developing sensitisation to dietary proteins, we prepared a plantlet extract without proteins and isolated 2 flavonoids, isoorientin-2''- O-arabinoside (1) and isovitexin-2''- O-arabinoside (2), and two saponins, avenacosides A (3) and B (4). The absence of protein in this extract was evidenced by electrophoresis and Western immunoblotting. Furthermore, Western immunoblotting demonstrated the absence of cross-reaction between grain and plantlet proteins. We evaluated the anti-inflammatory activity of the plantlet extract and its compounds IN VITRO in a model of keratinocyte inflammation: 6-keto prostaglandin F1 α production was inhibited by the plantlet extract (- 35 % and - 57 % at 10 and 30 µg/mL, respectively; p < 0.001) and isoorientin-2''- O-arabinoside (- 31 %, - 51 %, and - 56 % at 3, 10, and 30 µg/mL, respectively; p < 0.001). Intracellular interleukin-2 production in activated T lymphocytes was also inhibited by 16 %, 27 %, and 31 % with 3, 10, and 30 µg/mL plantlet extract, respectively, and by 23 % and 32 % with 3 and 10 µg/mL avenacoside A, respectively, (p < 0.001), demonstrating their immunoregulatory activity IN VITRO. The plantlet extract was also effective on the phenotype and function of dendritic cells (DC) differentiated from monocytes. It decreased the expression of major histocompatibility complex class II molecules on DC and significantly impaired their stimulatory activity on autologous T-cell proliferation (-25 %, p < 0.05). In conclusion, this protein-free oat plantlet extract exhibits anti-inflammatory and immunoregulatory activities in vitro.

BackgroundAtopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions resulting from structural and immune defects of the skin barrier. Previous studies have shown the clinical efficacy of Avène thermal spring water in AD, and a new microorganism, Aquaphilus dolomiae was suspected to contribute to these unique properties. The present study evaluated the anti-inflammatory, antipruritic, and immunomodulatory properties of ES0, an original biological extract of A. dolomiae, in immune and inflammatory cell models in order to assess its potential use in the treatment of AD.Materials and methodsAn ES0 extract containing periplasmic and membrane proteins, peptides, lipopolysaccharides, and exopolysaccharides was obtained from A. dolomiae. The effects of the extract on pruritus and inflammatory mediators and immune mechanisms were evaluated by using various AD cell models and assays.ResultsIn a keratinocyte model, ES0 inhibited the expression of the inflammatory mediators, thymic stromal lymphopoietin, interleukin (IL)-18, IL-4R, IL-8, monocyte chemoattractant protein-3, macrophage inflammatory protein-3α, and macrophage-derived chemokine and induced the expression of involucrin, which is involved in skin barrier keratinocyte terminal differentiation. In addition, ES0 inhibited protease-activated receptor-2 activation in HaCaT human keratinocytes stimulated by stratum corneum tryptic enzyme and T helper type (Th) 1, Th2, and Th17 cytokine production in Staphylococcal enterotoxin B–stimulated CD4+ lymphocytes. Lastly, ES0 markedly activated innate immunity through toll-like receptor (TLR) 2, TLR4, and TLR5 activation (in recombinant human embryonic kidney 293 cells) and through antimicrobial peptide induction (psoriasin, human beta-defensin-2, and cathelicidin), mainly through TLR5 activation (in normal human keratinocytes).ConclusionOverall, these in vitro results confirm the marked regulatory activity of this A. dolomiae extract on inflammatory and immune responses, which may be of value by virtue of its potential as an adjunctive treatment of AD inflammatory and pruritic lesions.

In addition to the extracellular production of O 2؊ by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b 559 , the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils. We examined whether these granules could be a site for intracellular production of O 2 ؊ . Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O 2 ؊ was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47 phox and p67 phox , two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47 phox and p67 phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O 2 ؊ was studied in retinoic aciddifferentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O 2 ؊ production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules. J. Leukoc. Biol. 65: 629-634; 1999.

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Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O − 2 producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O − 2 after 2 days of differentiation, they remain unable to generate O − 2 when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O − 2 in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.

SummaryIn 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.

The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression. Neurophysiology of painThe nociceptive system supports two sensory functions, pain and itch. Itch has often been regarded as a minor form of pain. Recently, it has been shown, however, that the pruritic system is supported by its own peripheral and central neuronal pathways which are closely associated, although antagonistic in some POMC processing in human melanocytes has been widely documented, and the a-MSH/MC1R/cAMP cascade has been implicated in the control of pigmentation. Only very recently, a role of b-endorphin, one cleavage product of b-LPH, has been demonstrated to influence melanocyte growth, dendricity and melanin biosynthesis via the m-opiate receptor. However, much earlier, it was shown that b-MSH, the other cleavage product of b-LPH, controls melanogenesis and melanin transfer in amphibians. To date, a specific receptor for b-MSH has not been identified. Earlier POMC processing has been found in melanosomes. Therefore, an MC1R-independent role of a-MSH was postulated and demonstrated in control of 6-tetrahydrobiopterin (6BH 4 )inhibited tyrosinase. Utilizing the depigmentation disorder vitiligo, we were now able to follow the fate of epidermal POMC processing in the presence of mM levels of hydrogen peroxide (H 2 O 2 ). In vitiligo epidermal PC2 and 7B2 protein expression is increased, whereas a-MSH, b-MSH and b-endorphin are significantly decreased. Analys...

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