EGFR-dependent aerotaxis is a common trait of breast tumour cells.

BACKGROUND: Aerotaxis, the chemotactism to oxygen, is well documented in prokaryotes. We previously reported for the first time that non-tumorigenic breast epithelial cells also display unequivocal directional migration towards oxygen. This process is independent of the hypoxia-inducible factor (HIF)/prolyl hydroxylase domain (PHD) pathway but controlled by the redox regulation of epidermal growth factor receptor (EGFR), with a reactive oxygen species (ROS) gradient overlapping the oxygen gradient at low oxygen concentration. Since hypoxia is an acknowledged hallmark of cancers, we addressed the putative contribution of aerotaxis to cancer metastasis by studying the directed migration of cancer cells from an hypoxic environment towards nearby oxygen sources, modelling the in vivo migration of cancer cells towards blood capillaries. METHODS: We subjected to the aerotactic test described in our previous papers cells isolated from fresh breast tumours analysed by the Pathology Department of the Saint-Etienne University Hospital (France) over a year. The main selection criterion, aside from patient consent, was the size of the tumour, which had to be large enough to perform the aerotactic tests without compromising routine diagnostic tests. Finally, we compared the aerotactic properties of these primary cells with those of commonly available breast cancer cell lines. RESULTS: We show that cells freshly isolated from sixteen human breast tumour biopsies, representative of various histological characteristics and grades, are endowed with strong aerotactic properties similar to normal mammary epithelial cell lines. Strikingly, aerotaxis of these primary cancerous cells is also strongly dependent on both EGFR activation and ROS. In addition, we demonstrate that aerotaxis can trigger directional invasion of tumour cells within the extracellular matrix contrary to normal mammary epithelial cells. This contrasts with results obtained with breast cancer cell lines, in which aerotactic properties were either retained or impaired, and in some cases, even lost during the establishment of these cell lines. CONCLUSIONS: Altogether, our results support that aerotaxis may play an important role in breast tumour metastasis. In view of these findings, we discuss the prospects for combating metastatic spread. TRIAL REGISTRATION: IRBN1462021/CHUSTE.

Inducible costimulator (ICOS) is a marker for highly suppressive antigen-specific T cells sharing features of TH17/TH1 and regulatory T cells.

BACKGROUND: CD4(+)CD25(+) regulatory T (Treg) cells are involved in the downmodulation of numerous immune responses to pathogens, tumors, or allergens. OBJECTIVE: In this study, we further characterized the nature of Treg cells that control skin inflammatory reactions to haptens. METHODS: In a model of contact hypersensitivity to 2,4-dinitro-fluorobenzene, we have investigated the phenotype, the specificity, and the origin of Treg cells that modulate the priming of effector CD8(+) T cells responsible for the development of the pathology. RESULTS: 2,4-Dinitrofluorobenzene immunization induced a population of CD4(+)CD25(+) Treg cells that controlled CD8(+) T-cell effector responses in a hapten-specific manner in vivo. High levels of inducible costimulator (ICOS) expression defined a population of CD4(+)CD25(+)FoxP3(+) (forkhead box protein 3) Treg cells that presented superior suppressive activity. Importantly, ICOS(+) Treg cells were distinguishable from all other FoxP3(+) Treg cells by the expression of IL-10, IL-17, and IFN-gamma. Hapten-specific Treg cells proliferating in response to their cognate antigen in vivo predominantly displayed a CD25(+)FoxP3(+)ICOS(+) phenotype. By using reporter mice, we showed that ICOS(+) Treg cells derived from the expansion of natural CD4(+)FoxP3(+) Treg cells rather than generation of adaptive Treg cells. Furthermore, the generation of ICOS(+) Treg cells depended on innate cells rather than the effector CD8(+) T-cell population. CONCLUSION: Taken together, our data show that a population of CD4(+)CD25(+)FoxP3(+) T cells upregulates ICOS on in vivo sensitization and specifically suppresses hapten-reactive CD8(+) T cells both in vivo and in vitro.

Patch testing in atopic dermatitis patients.

The exposure of atopic dermatitis (AD) patients to aeroallergens or food allergens can exacerbate or maintain the disease. Atopy patch tests (APTs) are able to identify these triggering factors and consist of the epicutaneous application of allergens for 48 hours, with an evaluation of the eczematous lesions induced after 48 and 72 hours, according to the reading criteria of the European Task Force on Atopic Dermatitis (ETFAD). APTs show a higher specificity than skin prick and specific IgE tests, since the pathophysiological mechanism of the reaction induced is very similar to that which occurs in AD lesions. The standardization of APTs to aeroallergens has brought a certain reliability to this method, which is not the case for food APTs, in which the positive predictive value must be improved to avoid any unnecessary dietary restrictions. Thus, optimization of APTs and progress in the knowledge of the pathophysiology of eczemas could help to develop new immunobiological diagnostic methods and specific immunotherapy for AD.

Allergic and irritant contact dermatitis.

Irritant and allergic contact dermatitis are common inflammatory skin diseases induced by repeated skin contact with low molecular weight chemicals, called xenobiotics or haptens. Although both diseases may have similar clinical presentations, they can be differentiated on pathophysiological grounds. Irritant contact dermatitis (ICD) is a non-specific inflammatory dermatitis brought about by activation of the innate immune system by the pro-inflammatory properties of chemicals. Allergic contact dermatitis (ACD) corresponds to a delayed-type hypersensitivity response with a skin inflammation mediated by hapten-specific T cells. Recent progress in the pathophysiology of chemical-induced skin inflammation has shown that ICD and ACD are closely associated and that the best way to prevent ACD is to develop strategies to avoid ICD. The immunological diagnosis of ICD or ACD requires investigation of the presence (ACD) or absence (ICD) of antigen-specific T cells. The detection of T cells can be performed in the skin (collected from ACD lesions or positive patch tests) and/or in the blood, particularly by using the enzyme-linked immunospot assay (ELISPOT). This method, recently developed in ACD to metals, offers a new biological tool enabling the immunobiological diagnosis of ACD.

Skin exposure to weak and moderate contact allergens induces IFNgamma production by lymph node cells of CD4+ T-cell-depleted mice.

Allergic contact dermatitis (ACD) is mediated by hapten-specific CD8+ T cells and downregulated by CD4+ T cells. We have recently shown in a model of ACD to weak haptens that priming of IFNgamma-producing CD8+ T cells and the development of skin inflammation could be obtained in mice deficient in CD4+ T cells. Here we show that IFNgamma production by lymph node (LN) cells draining the site of skin sensitization of CD4+ T-cell-deficient mice is a marker of the sensitizing properties of weak haptens. LN cells from mice sensitized as in the classical local lymph node assay (LLNA) were recovered at day 5, then cultured for 20 hours in the presence of submitogenic doses of phytohemagglutinin, and finally tested for the production of IFNgamma. Results show that: (i) production of INFgamma by LN cells was induced by weak and moderate allergens in a dose-dependent fashion; (ii) the magnitude of IFNgamma production paralleled the sensitizing properties of allergens allowing to classify them as moderate or weak haptens; (iii) chemicals without sensitizing properties were unable to stimulate IFNgamma production by LN cells. Therefore, the IFNgamma LLNA appears as a sensitive, specific, and robust assay to detect weak contact allergens.

Lack of evidence for allergenic properties of coumarin in a fragrance allergy mouse model.

BACKGROUND: There is controversy as to whether coumarin, an ingredient in cosmetics and fragrances, is a contact allergen involved in fragrance allergy. We recently showed that the purity of coumarin is a critical parameter for its allergenicity because coumarin preparations containing trace amounts of contaminants induced cell proliferation in the local lymph node (LN) assay whereas pure coumarin did not. OBJECTIVE/METHOD: In the present study, we analyzed the sensitizing properties of coumarin (purity > 99.9) and of dihydrocoumarin (DHC), in a recently developed model of fragrance allergy in mice. RESULTS: DHC was able to prime T cells in LNs draining the sensitization skin site and to induce a typical allergic contact dermatitis (ACD) reaction upon challenge, confirming that DHC is endowed with moderate sensitizing properties. In contrast, no T-cell activation and no ACD responses were obtained following sensitization and challenge with coumarin. CONCLUSION: These results confirm that pure coumarin is endowed with very weak sensitizing capacities, if any, and suggest that the presence of contaminants in coumarin preparations may account for the previously reported allergenic properties of coumarin.

Echographic measurement of skin thickness in adults by high frequency ultrasound to assess the appropriate microneedle length for intradermal delivery of vaccines.

Skin thickness (epidermis-dermis) across the deltoid, suprascapular, waist and thigh as possible body sites for a new microdelivery system for intradermal (id) inoculation were evaluated using 20 MHz ultrasound echography in 205 women and 137 men aged 18-70 years, in three ethnic groups: Caucasian, Asian and Black. Mean skin thickness was 2.54 mm at the suprascapular, 2.02 mm at the deltoid, 1.91 mm at the waist and 1.55 mm at the thigh. A 1.5 mm microneedle length inserted perpendicularly to the skin surface would ensure the administration of the antigen into the dermal layer, irrespectively of subject gender, age, ethnicity and BMI. The deltoid, suprascapular and waist are the most appropriate body sites.

Improvement of atopic dermatitis skin symptoms by Vitreoscilla filiformis bacterial extract.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease. The first line treatment of AD relies on the daily use of emollients to restore the skin barrier impairment associated with the disease. Vitreoscilla filiformis (V.f.) is a non photosynthetic bacterium and extracts of V.f. are endowed with properties which balance cutaneous immune-homeostasis. The aim of our study was to investigate the efficacy and safety of a 5% V.f. extract-containing ointment on mild to moderate AD in a randomised, double-blind, vehicle-controlled trial. Thirteen patients applied the treatment and the vehicle on symmetrical AD lesions (left versus right side of the body) twice daily for 4 weeks. The assessment of AD severity was done at each visit (Day 0, Day 14 and Day 28) using the modified eczema area and severity index (mEASI). Treatment with the ointment containing 5% V.f. extract significantly improved the AD skin symptoms. Beneficial effects were observed after two weeks of treatment and increased thereafter. These results suggest that V.f. extract could be favourably added to AD skin care emollients formulated for AD.

[Epidemiology and pathophysiology of eczemas]. / Approche epidémiologique et physiopathologique de l'eczéma atopique et de l'eczéma de contact.

Pathophysiological characteristics of these two types of eczema, both involve antigen specific effector T lymphocytes and down-regulatory CD4+ T cells (Treg). These cells are responsible for the cutaneous inflammatory response through the production of inflammatory cytokines and the destruction of keratinocytes by apoptosis. The main difference between these two types of disease is the nature of the environmental antigen. Indeed, protein allergens are involved in cases of atopic dermatitis whereas non protein chemicals (calles haptens) are responsible for allergic contact dermatitis. The skin lesions of eczema follow the activation of antigen specific T lymphocytes within the skin, leading to the production of inflammatory cytokines and to the destruction of keratinocytes by apoptosis. CD4+ Treg cells are endowed with down-regulatory and tolerogenic functions since they limit the skin inflammation in patients and prevent sensitization and induction of eczema in normal individuals. Eczema should therefore be considered as inflammatory dermatoses caused by the loss of immune tolerance to environmental antigens. Different strategies capable of restoring immune tolerance could avoid eczema outbursts and/or induce prolonged remissions of the disease.

Methotrexate for the treatment of adult atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease mediated by allergen-specific T cells which are recruited and activated in lesional skin. Methotrexate (MTX) is an old systemic agent used at low dosage for the treatment of psoriasis, another T cell-mediated skin disorder. Since MTX has been shown to improve the clinical symptoms of eczema in a model of antigen-specific dermatitis in mice, we postulated that it could be an effective treatment of AD. In the present open retrospective study, we report our results on the treatment of moderate to severe AD by MTX. Twenty patients (17 to 68-years-old) with low responses to routine therapies were treated (three months to 2 1/2 years) with a weekly dose of MTX ranging from 7.5 to 25 mg. The evaluation was made on physician's global assessment after 3 months of MTX use, and showed that 75% (15/20) of patients improved after 3 months of MTX use, among which 13/20 with an improvement>70%. The beginning of improvement was observed between the fourth and the eighth week after MTX was initiated. Tolerance was good. However, nausea and increase of liver enzymes were observed in 5 patients and required discontinuation of MTX in 2 patients. In conclusion, MTX seems to be an effective and safe treatment of AD. Placebo-controlled clinical trials are needed to confirm our observations and to define more precisely the effectiveness and safety of MTX in adult AD.

CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice.

Allergic contact dermatitis (ACD) to strong experimental haptens is mediated by specific CD8+ T cells. Here, we show that similar mechanisms occur for weak haptens, which comprise the vast majority of chemicals responsible for human ACD. We used a model of ACD, that is, the contact hypersensitivity reaction, to test for the allergenicity of three weak haptens involved in fragrance allergy. ACD to weak haptens could not be induced in normal mice. In contrast, mice acutely depleted in CD4+ T cells developed a typical ACD reaction to the three weak fragrance allergens that peaked 24 hours after challenge. Priming of CD8+ T cells was observed in draining lymph nodes 5 days after sensitization and development of ACD was associated with the infiltration of activated CD8+ T cells in the challenged skin. CD8+ T cells were effectors of the ACD reaction as in vivo treatment with depleting anti-CD8 mAbs abrogated the ACD responses and as purified CD8+ T cells could adoptively transfer ACD to naive recipients. In conclusion, our data demonstrate a dominant role of CD8+ T cells as initiators of ACD to weak haptens, and suggest that CD8+ T cells may represent potential targets for preventing or treating ACD.

[Allergic contact dermatitis: how to re-induce tolerance?]. / Eczéma allergique de contact: comment ré-induire une tolérance?

Allergic contact dermatitis (ACD) is a skin inflammatory disease mediated by activation of CD8+ cytotoxic T cells specific for haptens in contact with the skin. CD4+ T cells behave as both regulatory and tolerogenic cells since they down-regulate the skin inflammation in patients with ACD (regulation) and prevent the development of eczema (tolerance) in normal individuals. Thus, ACD corresponds to a breakdown of immune tolerance to haptens in contact with the skin. Several regulatory CD4+ T cell subsets (Treg), especially CD4+CD25+ natural Treg cells, are involved in immunological tolerance and regulation to haptens through the production of the immunosuppressive cytokines IL-10 and TGF-beta. Ongoing strategies to re-induce immune tolerance to haptens in patients with eczema include improvement of existing methods of tolerance induction (oral tolerance, low dose tolerance, allergen-specific immunotherapy, UV-induced tolerance) as well as development of new drugs able to activate IL-10 producing Treg cells in vivo. Ongoing and future progress in this area will open up new avenues for treatment of eczema and more generally autoimmune and allergic diseases resulting from a breakdown of tolerance to autoantigens and allergens, respectively.