Lack of functionally active Melan-A(26-35)-specific T cells in the blood of HLA-A2+ vitiligo patients.

Vitiligo, a skin disorder characterized by the spontaneous destruction of melanocytes, is believed to be of autoimmune origin. We investigated the presence and functionality of CD8(+) T-cells specific for the melanocyte-associated antigens Melan-A, gp100, tyrosinase, and TRP-2 in the blood of HLA-A2(+) vitiligo patients. We enumerated antigen-specific CD8(+) T cells by major histocompatibility complex multimer staining directly ex vivo, as well as after 9 days of in vitro stimulation and assessed IFN-gamma secretion by enzyme-linked immunospot (Elispot) assay. Tyrosinase-, gp100-, or TRP-2-specific CD8(+) T cells could not be identified in the peripheral blood of individuals with vitiligo. Although Melan-A-specific T cells were detectable at levels comparable to Flu-MP-specific T cells by multimer staining, these lymphocytes did not express the skin-homing receptor cutaneous lymphocyte antigen, were phenotypically naïve (CD45RA(+)), and were unresponsive in the IFN-gamma Elispot assay, suggesting that they are unlikely to be involved in the etiopathogenesis of vitiligo.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Dose-dependent effect of atrial natriuretic peptide on blood pressure, heart rate, and skin blood flow of normal volunteers.

Synthetic atrial natriuretic peptide, containing 26 amino acids in the rat sequence, L-364, 343 (Ileu-ANP), was infused intravenously at increasing rates (1-40 micrograms/min) into four normal volunteers. Mean intraarterial blood pressure decreased and heart rate increased in cumulative-dose-dependent fashion. Skin blood flow as measured with a laser Doppler device rose already with a cumulative dose of 55 micrograms Ileu-ANP and further rises were directly related to dose. The only side effects observed were those accompanying symptomatic hypotension at higher doses. These findings provide strong evidence that Ileu-ANP acts as a vasodilator in normal volunteers.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Online teaching of inflammatory skin pathology by a French-speaking international university network

Introduction: Developments in technology, webbased teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media like radiologic images, videos, clinical and macroscopic photographs and whole slides imaging is now accessible to almost every university. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of work, time and resources needed. In this perspective, a French national university network was initiated in 2011 to build mutualised online teaching pathology modules with clinical cases and tests. This network has been extended to an international level in 2012-2014 (Quebec, Switzerland and Ivory Coast). Method: One of the first steps of the international project was to build a learning module on inflammatory skin pathology intended for interns and residents of pathology and dermatology. A pathology resident from Quebec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform (http: //moodle.sorbonne-paris-cite.fr) under the supervision of two dermatopathologists (BV, MB). The learning module contains text, interactive clinical cases, tests with feedback, whole slides images (WSI), images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers. Results: The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 WSI and more than 50 micro and clinical photographs. The whole learning module is currently being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in spring 2014. The experience and knowledge gained from that work will be transferred to the next international fellowship intern whose work will be aimed at creating lung and breast pathology learning modules. Conclusion: The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated, completed and its use and existence needs to be promoted by the different actors in pathology. Of the great benefits of that kind of project are the international partnerships and connections that have been established between numerous Frenchspeaking universities and pathologists with the common goals of promoting education in pathology and the use of technology including whole slide imaging. * The Moodle website is hosted by PRES Sorbonne Paris Cité, and financial supports for hardware have been obtained from UNF3S (http://www.unf3s.org/) and PRES Sorbonne Paris Cité. Financial support for international fellowships has been obtained from CFQCU (http://www.cfqcu.org/).

In vivo activation of melanoma-specific CD8(+) T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells.

Many new types of vaccines against infectious or malignant diseases are currently being proposed. Careful characterization of the induced immune response is required in assessing their efficiency. While in most studies human tumor antigen-specific T cells are analyzed after in vitro re-stimulation, we investigated these T cells directly ex vivo using fluorescent tetramers. In peripheral blood lymphocytes from untreated melanoma patients with advanced disease, a fraction of tumor antigen (Melan-A/MART-1)-specific T cells were non-naive, thus revealing tumor-driven immune activation. After immunotherapy with synthetic peptides plus adjuvant, we detected tumor antigen-specific T cells that proliferated and differentiated to memory cells in vivo in some melanoma patients. However, these cells did not present the features of effector cells as found in cytomegalovirus specific T cells analyzed in parallel. Thus, peptide plus adjuvant vaccines can lead to activation and expansion of antigen specific CD8(+) T cells in PBL. Differentiation to protective CD8(+) effector cells may, however, require additional vaccine components that stimulate T cells more efficiently, a major challenge for the development of future immunotherapy.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.

Foot skin blood flow following infrainguinal revascularization for critical lower limb ischemia.

INTRODUCTION: The aim of this study was to assess the blood flow in the feet before and after lower limb revascularization using laser Doppler imaging (LDI). METHODS: Ten patients with critical lower limb ischemia were prospectively enrolled from June to October 2004. All patients underwent successful unilateral surgical interventions including above-knee bypass, distal bypass and endarterectomy. Skin blood flow (SBF) over the plantar surface of both forefeet and heels was measured by LDI 24h before and 10 days after revascularization, expressed in perfusion units (PU), and reported as mean+/-SD. RESULTS: Measurements in the forefoot and heel were similar. Before revascularization mean SBF was significantly lower in the ischemic foot (130+/-71 PU) compared to the contralateral foot (212+/-68 PU), p<0.05. After revascularization a significant increase of the SBF in the forefoot (from 135+/-67 to 202+/-86 PU, p=0.001) and hindfoot (from 148+/-58 to 203+/-83, p=0.001) was observed on the treatment side. However, a large decrease of the SBF was seen in forefoot and hindfoot on the untreated side (from 250+/-123 PU to 176+/-83 and from 208+/-116 to 133+/-40, p=0.001, respectively). CONCLUSION: This study confirms the benefits of revascularization in patients with nonhealing foot lesions due to critical limb ischemia. A significant increase of the SBF was observed on the treatment side. However, an unexpected decrease was observed on the untreated side.

The anti-apoptotic role of PPARbeta contributes to efficient skin wound healing.

PPARalpha and PPARbeta are expressed in the mouse epidermis during fetal development, but their expression progressively disappears after birth. However, the expression of PPARbeta is reactivated in adult mice upon proliferative stimuli, such as cutaneous injury. We show here that PPARbeta protects keratinocytes from growth factor deprivation, anoikis and TNF-alpha-induced apoptosis, by modulating both early and late apoptotic events via the Akt1 signaling pathway and DNA fragmentation, respectively. The control mechanisms involve direct transcriptional upregulation of ILK, PDK1, and ICAD-L. In accordance with the anti-apoptotic role of PPARbeta observed in vitro, the balance between proliferation and apoptosis is altered in the epidermis of wounded PPARbeta mutant mice, with increased keratinocyte proliferation and apoptosis. In addition, primary keratinocytes deleted for PPARbeta show defects in both cell-matrix and cell-cell contacts, and impaired cell migration. Together, these results suggest that the delayed wound closure observed in PPARbeta mutant mice involves the alteration of several key processes. Finally, comparison of PPARbeta and Akt1 knock-out mice reveals many similarities, and suggests that the ability of PPARbeta to modulate the Akt1 pathway has significant impact during skin wound healing.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.