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.

Effective inhibition of Th17/Th22 pathway in 2D and 3D human models of psoriasis by Celastrol enriched plant cell culture extract.

BACKGROUND: Psoriasis is an immune-mediated inflammatory disease in which the Th17 pathway is mainly involved. Systemic interventions with biologics that specifically block the Th17 pathway are effective to treat severe psoriasis. However, for efficient topical treatment, small molecules are more suitable than antibodies to penetrate and target epidermal keratinocytes, the key players in psoriasis. Celastrol, a well-described triterpene, is present in low amounts in Tripterygium wilfordii roots. By using plant cell culture (PCC), we were able to boost Celastrol production in bioreactors. Here, we evaluated immune modulator effect of Celastrol enriched extract (CEE) in Th17/Th22 psoriasis induced in 2D and 3D human models in vitro in view of its dermatological usage. METHODS: Human CD4+ T cells (hCD4), Normal Human Epidermal Keratinocytes (NHEK), micro-epidermis and reconstructed human epidermis (RHE) were preincubated with CEE and reference controls. Then, hCD4 were stimulated by anti-[CD3/CD28] while others were stimulated by Th17/22 cytokines cocktails. Psoriasis biomarkers were assessed by ELISA (hCD4 and RHE), by RT-qPCR (NHEK) or by ICH/ELISA (micro-epidermis). RESULTS: In 2D stimulated models (hCD4 and NHEK), CEE dose dependently inhibited, respectively, the expression of Th17 cytokines and psoriasis induced biomarkers. In 3D models (RHE and micro-epidermis), IL-8 expression was significantly reduced (RHE) and native phenotype was restored by CEE (micro-epidermis). CONCLUSION: These results clearly showed that Th17/Th22 cytokines, main inflammatory parameters, and psoriasis associated key biomarkers were inhibited by CEE in both 2D and 3D human in vitro models. Therefore, skin homeostasis could be restored by these modulator effects. Moreover, this high added value CEE was obtained by an ecofriendly bioprocess in contrast to traditional roots extracts. This is the first time that a well-defined CEE immune modulator has been proposed for psoriasis adjuvant care to reduce inflammation.

Cutaneous sensitivity modulation by Aquaphilus dolomiae extract-G3 on in vitro models of neuro-inflammation.

BACKGROUND: Inflammatory skin disorders, including atopic dermatitis (AD), associated pruritus and sensitive skin, have a complex multifactorial pathogenesis including neurogenic inflammation involving the release in blood and skin of neurotransmitters such as substance P (SP). AIMS AND METHODS: In vitro models evaluated the effect of the original biological extract of Aquaphilus dolomiae extract-G3 (ADE-G3) on cutaneous neurogenic inflammation. RESULTS: ADE-G3 significantly inhibited SP-stimulated release of IL-1ß and TNF-α from normal human epidermal keratinocytes; significantly and dose-dependently inhibited SP-stimulated activation of human mast cells; significantly inhibited veratridine-stimulated release of SP from human sensory neurons; modulated expression of genes involved in lipid synthesis, innate immunity, corneocyte scaffolding and epidermal differentiation in a histamine-sensitized reconstructed human epidermis model; and, when applied topically to ex vivo human explants, inhibited IL-8 and histamine release. CONCLUSIONS: Topically applied ADE-G3, once formulated, may improve neuro-inflammation in patients with inflammatory skin disorders.

Additional pharmacological activity of I-modulia and generation of two newly designed extracts of Aquaphilus dolomiae culture for dermocosmetic actives.

Aquaphilus dolomiae (AD) is a unique isolate from Avène Thermal Spring Water. I-Modulia, the first biotech extract from culture of AD, was used as immune modulator in Th2 inflammatory models. In this short publication, firstly we describe generation of two AD de novo extracts specifically designed for repairing and for neuroinflammation modulation activities which will be described, respectively, in two other articles in this supplement. Finally, for I-modulia, we describe new data on inhibition of human mast cell degranulation in vitro and its effect on substance P-induced neurogenic inflammation on ex vivo human skin explants.

Activités immunomodulatrice, anti-inflammatoire, antiprurigineuse et tolérogénique induites par I-modulia®, un extrait issu de culture d'Aquaphilus dolomiae, dans les modèles pharmacologiques de dermatite atopique: Immunomodulatory, anti-inflammatory, anti-pruritus and tolerogenic activities induced by I-modulia®, an Aquaphilus dolomiae culture extract, in atopic dermatitis pharmacology models.

Atopic dermatitis (AD) is an inflammatory and pruritic dermatosis of multifactorial origin. Topical steroids are the first line treatment for severe AD however alternatives treatment are increasingly needed. A biological concentrate was elaborated from culture of an Avène aquatic microflora isolate namely Aquaphilus dolomiae. Numerous extracts were evaluated in relevant AD in vitro models with human keratinocytes. Among these extracts, a particular one I-modulia® was found to be remarkable in terms of pharmacological activities: innate immunity modulating by agonizing Toll like receptor (TLR)2, TLR4 and TLR5, induction of anti-microbial peptides, inhibition of cytokines characteristics of T helper (Th)1, Th2 and Th17 responses, inhibition of Protease-activated-receptor (PAR) 2 and Thymic-stromal-lymphopoeitin (TSLP) both being known to be upregulated in pruritus. Additionally, when human dendritic cells (DC) were stimulated in vitro by Staphylococcus aureus secretomes from AD children lesions, I-modulia® was capable to induce IL-10 secretion to activate regular T lymphocytes and rendered DC tolerogenic. I-modulia®, extract of biotech origin incorporated in emollient, displays anti-inflammatory, anti-pruritus activities, restores homeostasis immune and ameliorates AD in young infant.

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.

Characterization of a human epidermis model reconstructed from hair follicle keratinocytes and comparison with two commercially models and native skin.

OBJECTIVE: Outer root sheath (ORS) cells of human hair follicles are a readily available, non-invasive source of keratinocytes for epidermis reconstruction. The aim of this study was to characterize a model of epidermis reconstructed from ORS cells (ORS-derived model) and to evaluate its reproducibility, in comparison with native human skin and two marketed reconstructed skin models (model A, Episkin(®) and model B, Skinethic(®) ). METHODS: Cell morphology and tissue architecture of the three models were analysed histologically and proliferation and differentiation marker expression by immunohistochemistry and mRNA quantification. RESULTS: All models displayed the same general epidermal architecture as native epidermis, but with a thicker stratum corneum in models A and B. Compared with native epidermis, Ki67 was correctly localized in epidermal basal cells in all models, as K10 in suprabasal layers. In all skin models, transglutaminase 1 (TGM1) was prematurely expressed in suprabasal layers. However, this expression was only observed from the upper stratum spinosum in the ORS-derived model. In this model, filaggrin and loricrin were correctly located in the stratum granulosum. Filaggrin, involucrin, loricrin and TGM1 mRNAs (markers of keratinocyte terminal differentiation) were transcriptionally expressed in all models. In the ORS-derived model, transcriptional expression level was similar to that of native skin. CONCLUSION: ORS cell-based reconstructed epidermis is a valid and reproducible model for human epidermis and it may be used to evaluate the effects of active substances and cosmetic formulations.

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.

Avène Thermal Spring Water: an active component with specific properties.

Avène Thermal Spring Water (TSW) is a natural active component characterized by a low mineral content. In vitro experiments have demonstrated the effect of Avène TSW on membrane fluidity, its antiradical and anti-inflammatory properties, its effects on many mediators involved in the immune response and its stimulating effect on keratinocyte differentiation. The clinical efficacy of the water was demonstrated at the hydrotherapy centre in chronic and disabling diseases such as atopic dermatitis but also in various settings in medical and post dermatology procedure such as photodynamic therapy or photothermolysis. All these data support the fact that the Avène TSW is an active component.

Acceleration of keratinocyte differentiation by transient receptor potential vanilloid (TRPV6) channel activation.

BACKGROUND: Numerous studies have demonstrated the beneficial effect of Avène Thermal Spring Water (TSW) in dermatological diseases but the molecular mechanisms remain unknown. The objective of the present study was to evaluate the effect of Avène TSW on the morphological and molecular features related to the more advanced status of differentiation of human keratinocytes. MATERIAL AND METHODS: Normal human keratinocytes (NHK) were differentiated in medium powder reconstituted with Avène TSW and assessed by RT-PCR and immunohistochemistry. Calcium entry was measured by a Fura-2 AM probe. TRPV6 channel were detected by immunohistochemistry, RT-PCR and western blot. RESULTS: Treatment of NHK with Avène TSW led to an enhanced constitutive calcium entry that resulted in the increased expression of involucrin and cytokeratins 1 and 10. This enhanced constitutive calcium entry in Avène TSW-treated keratinocytes was mediated by the TRPV6 calcium channel. Moreover, Avène TSW-mediated calcium entry was due to the increase in TRPV6 expression as well as the channel abundance at the cell membrane. CONCLUSIONS: An other mechanism of action of Avène TSW is described. Avène TSW treatment induced an enhanced constitutive calcium entry mediated by TRPV6 channel leading to the acceleration of human keratinocytes differentiation.

Laparoscopic dorsal subsegmentectomy 8: Exploit the 3d technology to plan liver resection, and predict intraparenchymal pedicles. A case report. (With video explanation).

INTRODUCTION: Laparoscopic liver surgery is spreading, encouraged by technical and technological improvement. Both the obligated narrow space and the difficulty to modify it lead to a more complex approach to the lesions located in the posterosuperior portion of the liver. Surgical strategies such as the Caudal approach or the Diamond technique could ameliorate surgical procedure, but these areas remain a challenge and are still classified as complex. CLINICAL CASE: We discuss the case of a 68 year old man with metachronous liver metastasis in the dorsal part of segment 8. We used portal phase CT-scan Dicom data to create Three-dimensional reconstruction, which was able to show the more distal branches of intraparenchymal structures. The reconstructed images were subsequently used to plan laparoscopic liver resection. DISCUSSION: The capability of three-dimensional reconstruction to create a realistic image allows us to use ultrasound as a navigation tool. Exploiting these two technologies together, we arrived to regulate the resection stages by recognizing previously marked structures and searching them at every intervention phase. The strategy performed demonstrates both a high level of precision and the capability to predict intraparenchymal structures. CONCLUSION: The advantages obtained from three-dimensional reconstruction are numerous in terms of either anatomical comprehension and technical precision, suggesting a potential improvement in surgical skill. Three-dimensional technology should be encouraged and spread to understand, in every single aspect, the potential of its use.

Extracellular vesicles enhance the targeted delivery of immunogenic oncolytic adenovirus and paclitaxel in immunocompetent mice.

Extracellular vesicles (EVs), are naturally occurring cargo delivery tools with the potential to be used as drug vehicles of single agents or combination therapies. We previously demonstrated that human lung cancer cell-derived EVs could be used for the systemic delivery of oncolytic virus (OVs) and chemotherapy drugs such as paclitaxel (PTX), leading to enhanced anti-tumor effects in nude mice. In the current work, we evaluated the biodistribution of EVs by using bioluminescence and fluorescence imaging technologies, thus proving the ability of these EVs-formulations to specifically target the neoplasia, while leaving other body tissues unaffected. Moreover, in vivo imaging of NFκB activation in an immunocompetent reporter mouse model allowed to demonstrate the selective ability of EVs to induce tumor-associated inflammatory reactions, which are characterized by immunogenic cell death and CD3+/CD4+/CD8+ T-cell infiltration. While EVs have the potential to induce a systemic immune reaction by pro-inflammatory cytokines, our study provides compelling evidences of a localized inflammatory effect in the peritumoral area. Collectively, our findings strongly support the systemic administration of EVs formulations with OVs alone or in combination with chemotherapy agents as a novel strategy aimed at treating primary and metastatic cancers.

Endocrine influence on neuroinflammation: the use of reporter systems.

Most of the ageing-associated pathologies are coupled with a strong inflammatory component that accelerates the progress of the physiopathological functional decline related to ageing. The currently available pharmacological tools for the control of neuroinflammation present several side effects that restrict their application, particularly in chronic disorders. The discovery of the potential anti-inflammatory action exerted by endogenous oestrogens, as well as the finding that activation of oestrogen receptor α results in a significant decrease of inflammation at the cellular level and in models of inflammatory diseases, prompted us to embark in a series of studies aimed at the generation of reporter systems, allowing us to (i) understand the anti-inflammatory action of oestrogens at molecular level; (ii) evaluate the extent to which the action of this steroid hormone was relevant in models of pathologies characterised by a strong inflammatory component; and (iii) investigate the efficacy of novel, synthetic oestrogens endowed with anti-inflammatory activity. Accordingly, we conceived the NFκB-luc2 reporter mouse, a model characterised by dual reporter genes for fluorescence and bioluminescence imaging under the control of a synthetic DNA able to bind the transcription factor nuclear factor kappa B, the master regulator of the expression of most of the cytokines responsible for the initial phase of acute inflammation. Here, we summarise the philosophy that has driven our research in the past years, as well as some of the results obtained so far.

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.

FISH analysis in Prader-Willi and Angelman syndrome patients.

We report on a combined high resolution cytogenetic and fluorescent in situ hybridization study (FISH) on 15 Prader-Willi syndrome (PWS) and 14 Angelman syndrome (AS) patients. High resolution banding showed a microdeletion in the 15q11-q13 region in 7 out of 15 PWS patients, and FISH analysis of the D15S11 and SNRPN cosmids demonstrated absence of the critical region in three additional cases. Likewise 8 out of 14 AS patients were found to be deleted with FISH, using the GABRB3 specific cosmid, whereas only 4 of them had a cytogenetically detectable deletion.

FISH characterization of small supernumerary marker chromosomes in two Prader-Willi patients.

A small supernumerary chromosome was observed in two Prader-Willi syndrome (PWS) patients. The clinical diagnosis of PWS was confirmed by the ascertainment of the deletion of region 15q11-13 in one case and uniparental disomy (UPD) of the same region in the other. The markers were negative for dystamycinA/DAPI banding, did not contain NOR-positive satellites, and had an appearance consistent with a very small ring chromosome. Fluorescent in situ hybridization (FISH) analysis with the "all human centromere" probe indicated the presence of centromeric sequences in both markers. Chromosomal in situ suppression hybridization with chromosome specific libraries demonstrated that the small markers in the deleted and UPD patient originated from chromosome 15 and X, respectively. To the best of our knowledge these are the only PWS patients reported with a supernumerary marker chromosome other than inv dup(15) characterized by FISH.

FISH characterization of two supernumerary r(1) associated with distinct clinical phenotypes.

Only a few reports on supernumerary r(1) chromosomes associated with a clinical phenotype have been published. We describe two unrelated patients with congenital malformations and developmental delay who were found to have a de novo supernumerary r(1) in 50% (Case 1) and 80% (Case 2) of the examined cells. Conventional cytogenetic techniques (QFQ, CBG, and DA-DAPI), complemented by fluorescence in situ hybridization studies using alpha satellite probes, showed that both small marker chromosomes (SMCs) primarily consisted of the centromere and heterochromatin of chromosome 1, a conclusion that was also supported by chromosome 1 painting. In an attempt to establish phenotype-genotype correlations, a further investigation was performed using YACs mapped to the chromosome 1 pericentromeric region. A fluorescent signal was evident after hybridization with Y934G9 (1q21) in Case 1 and Y959C4 (1p11.1-12) in Case 2. Partial trisomy of unique sequences flanking pericentromeric sequences is shown to underlie the clinical phenotype in both patients. This evidence should be taken into account when SMCs are ascertained, particularly in prenatal diagnosis.

Cytogenetic abnormalities detected by direct analysis in a case of choriocarcinoma.

Malignant trophoblastic cells from a case of choriocarcinoma were cytogenetically investigated by direct analysis of fresh tissue from the tumor. To our knowledge, previous cytogenetic studies have been performed only on established cell lines. In this study, 54 metaphases were observed, of which 41 were fully karyotyped. Three chromosomally abnormal lines were identified. In all of them, trisomy 3 and 10, a supernumerary isochromosome 1q,i(1)(q10), an i(8)(q10) replacing one chromosome 8, and a marker chromosome were observed. In addition, involvement of chromosome 12 was observed in two of the three lines, trisomic in one and an i(12)(q10) in the other.