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Background: The regulation of melanoma by noradrenergic signaling has gain attention since pre-clinical and clinical studies suggested a benefit of using beta-blockers to control disease progression. We need to confirm that human melanoma recapitulates the mechanisms described from pre-clinical models. Methods: The sources and targets of norepinephrine in the microenvironment of 20 human melanoma samples was investigated using immunostaining. The effect of an exposure to beta-blockers on immune cell type distribution and expression of immune response markers was assessed with immunostaining on 212 human primary melanoma. A statistical analysis explored the effect of an exposure to beta-blockers on progression free survival, melanoma related survival, and overall survival on the 286 eligible patients. Results: Tumor cells and macrophages may be a source of norepinephrine in melanoma microenvironment. Tumors from patients exposed to wide spectrum beta-blockers recapitulate the increased infiltration of T-lymphocytes and the increased production of granzyme B observed in pre-clinical models. An exposure to beta-blockers is associated with a better outcome in our cohort of melanoma patients. Conclusion: This study shows the association between an exposure to wide spectrum beta-blockers and markers of an effective anti-tumor immune response as well as the protective effect of beta-blockers in human melanoma patients.

Betablockers are used for decades in the treatment of cardiovascular diseases. Recently, the repurposing of the wide spectrum betablocker propranolol revolutionized the infantile hemangioma management. Besides, betablockers have been shown to significantly improve the prognosis of melanoma patients. Blocking the adrenergic receptors blocks the stress-induced tumorigenic pathways. Propranolol inhibits melanoma by downregulating the tumor angiogenesis but also tumor cell proliferation, invasiveness and local immune suppression. These antitumor effects have been observed in other cancers. The repurposing of propranolol has already changed the practice of pediatricians and dermatologists and may soon help oncologists treat various cancers., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

In a previous study on a xenograft model of melanoma, we showed that the beta-adrenergic receptor antagonist propranolol inhibits melanoma development by modulating angiogenesis, proliferation and cell survival. Stress hormones can influence tumor development in different ways and norepinephrine was shown to downregulate antitumor immune responses by favoring the accumulation of immunosuppressive cells, impairing the function of lymphocytes. We assessed the effect of propranolol on antitumor immune response in the MT/Ret mouse model of melanoma. Propranolol treatment delayed primary tumor growth and metastases development in MT/Ret mice. Consistent with our previous observations in human melanoma xenografts, propranolol induces a decrease in cell proliferation and vessel density in the primary tumors and in metastases. In this immunocompetent model, propranolol significantly reduced the infiltration of myeloid cells, particularly neutrophils, in the primary tumor. Inversely, cytotoxic tumor infiltrating lymphocytes were more frequent in the tumor stroma of treated mice. In a consistent manner, we observed the same shift in the proportions of infiltrating leukocytes in the metastases of treated mice. Our results suggest that propranolol, by decreasing the infiltration of immunosuppressive myeloid cells in the tumor microenvironment, restores a better control of the tumor by cytotoxic cells.

Background: Lock-in thermal imaging is a thermographic method that is widely used in the nondestructive testing of materials. The technique allows detecting under the sample surface, small variations of the thermophysical properties in a noninvasive and noncontact manner. Surprisingly, this method has, to our knowledge, never been used in dermatology although it is particularly suited., Methods: We present in this article the first lock-in thermal imaging setup dedicated to dermatological applications. The apparatus uses a temperature-modulated airflow to periodically stimulate the skin surface. The infrared images recorded by a high sensitive camera are demodulated according to the digital lock-in principle to compute a phase and amplitude image., Results: First results obtained on benign skin lesions are presented. The images allow to detect small variations of the tissue thermophysical properties like for example, perfusion variations. Lock-in thermal imaging has the ability to reject disturbing thermal signals coming from subcutaneous tissues. The localization of the lesions is more accurate due the suppression of the lateral heat spreading., Conclusion: Lock-in thermal imaging is a promising method for the detection of lesions exhibiting modified thermophysical properties compared to the surrounding healthy skin., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recently, retrospective studies provided conflicting results on the benefit of β-adrenoceptor-blockers (β-blockers) on melanoma progression. Most of these studies did not define the β-blocker used, making it difficult to understand the source of discrepancies between results. Therefore, we investigated the effect of non-cardioselective and cardioselective β-blockers on melanoma progression at the cellular, molecular, and tumor levels. Here we show that the non-cardioselective β-blocker propranolol hydrochloride (propranolol) inhibits proliferation and induces apoptosis in primary cell cultures derived from a primary and a metastasis of human melanoma and in melanoma cell lines. In contrast, the cardioselective β-blocker metoprolol tartrate hardly affects melanoma cell survival or proliferation. We further highlight that a daily treatment with propranolol slows down tumor development in immunodeficient mice transplanted with human melanoma cells. RNA microarrays, quantitative PCR, and histochemistry analyses showed that propranolol regulates the expression of different genes involved in tumor angiogenesis, cell death, or proliferation. Thus, our results suggest that non-cardioselective β-blockers affect melanoma progression, and bring first clues about the pathways involved in this antitumor effect.

This paper theoretically evaluates lock-in thermal imaging for the early-stage detection of cutaneous melanoma. Lock-in thermal imaging is based on the periodic thermal excitation of the specimen under test. Resulting surface temperature oscillations are recorded with an infrared camera and allow the detection of variations of the sample's thermophysical properties under the surface. In this paper, the steady-state and transient skin surface temperatures are numerically derived for a different stage of development of the melanoma lesion using a two-dimensional axisymmetric multilayer heat-transfer model. The transient skin surface temperature signals are demodulated according to the digital lock-in principle to compute both a phase and an amplitude image of the lesions. The phase image can be advantageously used to accurately detect cutaneous melanoma at an early stage of development while the maximal phase shift can give precious information about the lesion invasion depth. The ability of lock-in thermal imaging to suppress disturbing subcutaneous thermal signals is demonstrated. The method is compared with the previously proposed pulse-based approaches, and the influence of the modulation frequency is further discussed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nevus of Ota is a unilateral, asymptomatic cutaneous and mucosal hyperpigmentation of the face that is congenital or may appear during childhood. We present a case of symptomatic acquired nevus of Ota in an adult, associated with intense pruritus, not described in the literature so far. A 32-year-old woman presented with brownish mottled macules which appeared on her face progressively over 8 days, following the distribution of the first and second divisions of the left trigeminal nerve and partially covering the iris and sclera of the left eye. She reported an intense pruritus in this area. We performed a biopsy on the left forehead, which confirmed the diagnosis of nevus of Ota. Specific stains and immunohistochemistry revealed increased numbers of mast cells. Ophthalmological tests showed acute acquired melanocytosis of the left iris and sclera. The origin of the nevus is still unclear. Several hypotheses suggest a reactivation of melanocytes during their migration from the neural crest. The pruritus reported in our patient may be explained by the increased quantity of mast cells observed in the lesion and/or neuronal stimulation of the ophthalmic and maxillary divisions of the fifth cranial nerve.

Background: Desmoplastic trichoepithelioma (DT) is a rare benign adnexal neoplasm considered to have follicular differentiation. It usually presents as an asymptomatic, firm, annular plaque with a raised border. The diagnosis of DT is based on clinical and histological features which can be similar to those of morpheiform basal cell carcinoma. Taking this into consideration, the use of another diagnostic technique would be very useful. Dermoscopy is a noninvasive diagnostic technique allowing a more accurate diagnosis., Objectives: To differentiate DT and morpheiform basal cell carcinoma by means of dermoscopy., Methods: Here, we describe the dermoscopic features of a case of DT, and make a direct clinicopathological correlation by using the horizontally cut slides provided by Mohs micrographic surgery., Results: On dermoscopy, DT shows well-defined borders and an ivory-white color, as well as prominent arborizing telangiectasias in the central area and on the right side. There are no leaf-like structures and no ovoid nests. This observation confirms and completes the first dermoscopic analysis of DT reported in the literature so far., (Copyright © 2013 S. Karger AG, Basel.)

Increased risk of secondary melanoma after breast cancer has been reported. Several lines of evidence suggest that elevated estrogen levels may be implicated in melanoma etiology. Accordingly, use of antiestrogens should be associated with decreased risk of melanoma. We compared melanoma incidence among a cohort of breast cancer patients with and without antiestrogen therapy, with data from the Geneva Cancer Registry. The cohort consisted of 7,360 women diagnosed with breast cancer between 1980 and 2005. About 54% of these patients received antiestrogens. All women were followed until December 2008. We compared cutaneous melanoma incidence rates among patients with and without antiestrogens with those expected in the general population by age and period standardized incidence ratios (SIR). A total of 34 women developed a melanoma during the follow-up period. Compared with the general population, the risk of melanoma was higher for patients who did not receive antiestrogens (SIR: 1.60, 95% CI: 1.08-2.12, P = 0.02). On the contrary, the risk was close to 1 (SIR: 0.98, 95% CI: 0.40-1.56, P = 0.57) for patients who received antiestrogen therapy. This study suggests that antiestrogen therapy modifies the risk of melanoma after breast cancer. Although our results are in agreement with the hypothesis that estrogens could play a role in melanoma occurrence, they need to be replicated in a larger study with data on potential confounders. ., (©2011 AACR)

Lentigo maligna (LM) and lentigo maligna melanoma (LMM) account for about 10% of all melanomas. They have distinct clinical and histological features as well as specific epidemiology, natural history, evolution, and genetics. Epidemiology of these tumors progressively concerns younger patients. Diagnostic and preparation of surgical removal of LM and LMM can now be optimized by new technologies of imaging, like dermoscopy or in vivo confocal reflectance microscopy. Surgery is the treatment of choice, but alternative options can be considered, especially for the elderly for whom the needs for efficiency and acceptability should be met.

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Background: Scleroedema adultorum Buschke (SB) is a rare disease involving scleroedema of the neck and shoulders. It can extend to the rest of the trunk and the limbs but characteristically spares the extremities. Three types of SB are distinguished: the first is acute and develops after an infectious disease, the second is of insidious evolution and is associated with monoclonal gammopathy, and the third is associated with type 2 diabetes., Patients and Methods: We report the case of a type 2 diabetic patient presenting with progressive, oedematous timbering of the trunk associated with impaired mobility, dysphagia and restrictive respiratory syndrome. SB was diagnosed on the basis of clinical presentation and histology. Treatment was mandatory because of the adverse impact of the disease. A therapy that would not worsen the patient's comorbidities had to be chosen. Intravenous immunoglobulins were thus initiated with excellent response as of the first cycle regarding trunk mobility and dysphagia. Cutaneous rigidity improved steadily until the end of treatment (eight cycles)., Conclusion: Therapeutic abstention is the rule in SB if it has no severe functional repercussions. Nevertheless, there is no clearly indicated treatment once therapy becomes necessary. Control of underlying diabetes usually does not improve the scleroedema and the metabolic syndrome contraindicates most of the treatments reported in the literature. In this article, we suggest a new treatment of SB in the diabetic patient.

Due to the early diagnosis, melanomas can be diagnosed in early stages. Most melanomas tend not to show morphological criteria of malignancy in the very early stages. They rather resemble benign moles. For patients with hundreds of atypical lesions, follow-up examinations using digital dermoscopy are very helpful. This technique enables the physician to monitor lesions and to detect microscopic change. Lesions with microscopic change are thought to be high risk lesions and should be removed this will represent important savings for the health system because this will allow to make the diagnosis of melanoma in earlier stages and to save costs for unnecessary surgery. In this article we are going to review the technique.

Longitudinal pigmentation of the nail is very common. The differential diagnosis varies from subungual hematoma, to a fungal infection, to a melanocytic lesion (lentigo, nevus melanoma, etc.) to others. Often, dermatologists do not feel at ease with these pathologies and management is often not clear. In many cases, a biopsy is not helpful because an inadequate technique was chosen. The use of noninvasive techniques such as dermoscopy has been described to be useful for the preoperative evaluation and the management decision. Using these technique, one will be able to reduce the number of unnecessary surgeries and to choose the most adequate biopsy technique. In this article, we will review the management, including diagnosis as well as differential diagnosis of nail pigmentations and propose a new algorithm for the non invasive diagnosis of nail pigmentation.

Tumor antigen-specific cytotoxic T cells (CTLs) play a major role in the adaptive immune response to cancers. This CTL response is often insufficient because of functional impairment, tumor escape mechanisms, or inhibitory tumor microenvironment. However, little is known about the fate of given tumor-specific CTL clones in cancer patients. Studies in patients with favorable outcomes may be very informative. In this longitudinal study, we tracked, quantified, and characterized functionally defined antigen-specific T-cell clones ex vivo, in peripheral blood and at tumor sites, in two long-term melanoma survivors. MAGE-A10-specific CD8+ T-cell clones with high avidity to antigenic peptide and tumor lytic capabilities persisted in peripheral blood over more than 10 years, with quantitative variations correlating with the clinical course. These clones were also found in emerging metastases, and, in one patient, circulating clonal T cells displayed a fully differentiated effector phenotype at the time of relapse. Longevity, tumor homing, differentiation phenotype, and quantitative adaptation to the disease phases suggest the contribution of the tracked tumor-reactive clones in the tumor control of these long-term metastatic survivor patients. Focusing research on patients with favorable outcomes may help to identify parameters that are crucial for an efficient antitumor response and to optimize cancer immunotherapy.

A PRoliferation-Inducing TNF Ligand (APRIL) costimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia, reminiscent of human B-cell chronic lymphoid leukemia (B-CLL). We analyzed APRIL expression in situ in human non-Hodgkin lymphomas. APRIL up-regulation was only observed in high-grade B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL). Up-regulation was seen in 46% and 20% of DLBCL and BL, respectively. In DLBCL, neutrophils, constitutively producing APRIL and infiltrating the tumor tissue, were the main cellular source of APRIL. Rare DLBCL cases showed a predominance of histiocytes or mesenchymal cells as APRIL source. APRIL secreted by neutrophils accumulated on tumor cells via proteoglycan binding. In addition to proteoglycans, DLBCL tumor cells expressed the APRIL signaling receptor, TACI and/or BCMA, indicating that these tumor cells are fully equipped to respond to APRIL. A retrospective clinical analysis revealed a significant correlation between high expression of APRIL in tumor lesions and decreased overall patient survival rate. Hence, APRIL produced by inflammatory cells infiltrating lymphoma lesions may increase tumor aggressiveness and affect disease outcome.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Spontaneous immune responses to the cancer testis antigen NY-ESO-1 are frequently found in cancer patients bearing antigen-expressing tumors. In HLA-A2-expressing patients, naturally elicited NY-ESO-1-specific, tumor-reactive cytotoxic T lymphocytes (CTLs) are mostly directed against an immunodominant epitope corresponding to peptide NY-ESO-1 157-165. NY-ESO-1-specific CTLs can also be induced by synthetic peptide vaccines, but they are heterogeneous in terms of functional avidity and tumor reactivity. The authors investigated the structural bases of this phenomenon by analyzing the TCR features of natural and vaccine-induced NY-ESO-1-specific CTLs. The results indicate that CTLs from the two groups exhibit highly structurally conserved but distinct TCR features, suggesting that the synthetic peptides used for vaccination may fail to faithfully mimic the naturally processed antigen. Together, the results of this study underline the strength of TCR molecular monitoring and will be instrumental for the development and monitoring of vaccines aimed at eliciting CTLs with high tumor reactivity.

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.

Tumor antigen-reactive T cells can be detected in a large proportion of melanoma patients, but their efficacy on tumor control in vivo remains unclear. On the other hand, vitiligo, a skin disorder characterized by patchy depigmented macules, may occur spontaneously or after antitumor therapies. Moreover, vitiligo is significantly associated with positive clinical response, but the mechanism is not understood. Therefore, the establishment of a relevant animal model in which melanoma and vitiligo spontaneously develop stepwise may be useful for better understanding of the parameters involved in the destruction of both benign and malignant melanocytes. In a previous work, we established a mouse model for melanoma in which MT/ret transgenic mice express the ret oncogene fused to the metallothionein promoter. Here we report that melanoma leads to spontaneous vitiligo. We further investigate, for the first time in this model, the natural antitumor T-cell response and evaluate the role of cellular immunity in the development of the disease. Interestingly, the occurrence of spontaneous tumor nodules in MT/ret mice with melanoma-associated vitiligo is significantly delayed when compared in melanoma mice without vitiligo. Moreover, a significant proportion of mice with melanoma-associated vitiligo resisted a challenge with syngeneic melanoma cells in contrast to animals without vitiligo. Our results confirm that vitiligo is associated with clinical benefit and further demonstrate the crucial role of CD8+ T cells for tumor control in melanoma-associated vitiligo.

We described a new process for the design of HLA tetramers using soluble MHC class I molecules purified from Epstein Barr Virus-transformed B cells. This method does not rely on genetic engineering and presents a significant advantage in view of the polymorphism of MHC class I molecules because tetramers can be produced with any HLA molecule. Here, we showed that our HLA-A*0201 tetramers provided experimental results similar to those obtained with tetramers made with recombinant MHC molecules. Moreover, they can be used to efficiently identify peptide-specific T cells from ex vivo PBMCs as well as from lymphocytes infiltrating human tumor. This innovative and simple method could be widely adopted, specially in diagnostic procedures for monitoring peptide-based immunotherapy.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Identification of tumor antigens and their optimal antigenic peptides raised hopes for the development of peptide-based immunotherapeutic vaccine strategies for human melanoma, however. Synthetic peptides alone are not immunogenic enough, and adequate formulation is critical for elaboration of peptide vaccines. To improve formulation, we evaluated 2 lipopeptide constructs, both including HLA-A2-restricted MART 27-35-CD8+ T lymphocyte (CTL) epitope covalently linked to universal tetanus toxoid (TT) 830-843 helper T lymphocyte (HTL) epitope, in HLA-A2 transgenic mouse models that mimic human CTL responses in vivo. These 2 constructs only differed in the formulation of their lipid tail. We showed that lipopeptide constructs were strongly recognized, in vitro, by human MART 27-35 cytotoxic T cells derived from tumor-infiltrating lymphocytes. The transgenic Mice immunized with these 2 MART lipopeptide formulations containing covalently linked HTL-CTL epitopes induced strong MART 27-35 cytotoxic T cells. This CTL induction was critically dependent on the presence of the helper T lymphocyte epitope. These results also showed that a single palmitoyl-lysine chain is enough to assure immunogenicity of a given peptide and that the presence of a lipid tail bypass the need for adjuvant. These results support the selection of MART-lipopeptide melanoma vaccine for evaluation in a clinical trial., (Copyright 2001 Wiley-Liss, Inc.)

Vitiligo is a cutaneous pigmentary disorder characterized by the loss of melanocytes. An autoimmune mechanism is strongly suspected to be involved in this affection given that it is frequently associated with autoimmune hormonal disorders, and because antibodies directed against melanocytic antigens are found in the serum of patients with vitiligo. We examined the role of cellular immunity in melanoma-associated vitiligo by expanding infiltrating lymphocytes from fresh biopsy specimens of vitiligo patches in melanoma patients. The vitiligo-infiltrating lymphocytes were almost exclusively T lymphocytes, and most were CD8(+). Following in vitro expansion, vitiligo-infiltrating lymphocytes remained predominantly CD8(+) and expressed the cutaneous homing receptor CLA. Furthermore, vitiligo-infiltrating lymphocytes had a clonal or oligoclonal T cell receptor profile, possibly reflecting specific antigenic stimulation. Finally, vitiligo- infiltrating lymphocytes specifically recognized differentiation antigens shared by normal melanocytes and melanoma cells. This direct demonstration of CD8(+) T cell involvement in vitiligo suggests that, in melanoma patients, vitiligo may be a visible effect of a spontaneous antitumoral immune response.

The design of heteroclytic antigens with high MHC binding capacity is of particular interest to overcome the weak immunogenicity of peptide epitopes derived from tissue antigens expressed by tumors. In the present study, double-substituted peptide analogues of the tumor-associated antigen MART-1(27-35) incorporating a substitution at a primary anchor residue and a beta-amino acid residue at different positions in the sequence were synthesized and evaluated for binding to the human histocompatibility class I molecule HLA-A2 and for recognition by tumor-infiltrating lymphocytes. Interestingly, by combining a Leu for Ala substitution at P2 (which alone is deleterious for antigenic activity) with a beta-amino acid substitution at a putative TCR contact residue, recognition by tumor-infiltrating lymphocytes was partially restored. The analogue [Leu(28),beta-HIle(30)]MART-1(27-35) displays both a higher affinity to HLA-A2 and a more prolonged complex stability compared to [Leu(28)]MART-1(27-35). Overall, these results suggest that double-substitution strategies and beta-amino acid replacements at putative TCR contact residues might prove useful for the design of epitope mimics with high MHC binding capacity.

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.

In the present study, we demonstrate that the non-classical MHC class I molecule HLA-G impairs specific cytolytic T cell functions in addition to its well-established inhibition of NK lysis. The antigen-specific cytotoxic T lymphocyte (CTL) response analyzed was mediated by CD8(+) T cells specific for the influenza virus matrix epitope, M58-66, presented by HLA-A2. The transfection of HLA-G1 cDNA in target cells carrying the M58-66 epitope reduced their lysis by these virus-specific CTL. This HLA-G-mediated inhibition of antigen-specific CTL lysis was (i) peptide dose dependent, (ii) reversed by blocking HLA-G with a specific mAb and (iii) still observed despite the blockade of HLA-E/CD94/NKG2A interaction. By inhibiting both CTL and NK functions, HLA-G appears to have an extensive role in immune tolerance.

Nonclassical MHC class I HLA-G antigen expression is tissue specific and is thought to play a role in tolerance of the semiallogeneic fetus by the maternal immune system. Ectopic expression of HLA-G by tumor cells provides them with an additional mechanism of escape from immunosurveillance by host cytotoxic effector mechanisms. The aim of this study was to assess the expression of nonclassical HLA-G antigens in ex vivo human melanoma biopsies. HLA-G mRNA levels corresponding to both membrane-bound and soluble protein isoforms were analyzed in tumor specimens obtained from primary or metastatic melanomas of 23 patients. High levels of HLA-G transcription were detected in tumor specimens in 5 of 23 patients and found to be comparable in both lymph node and skin metastases. HLA-G mRNA transcript levels at tumor sites in 18 of these patients were compared with those in samples of their own healthy skin and were higher in the tumor tissue in 12 patients. Differential expression of mRNA transcripts corresponding to soluble and membrane-bound HLA-G was also observed in some tumor biopsies. HLA-G protein expression was detected in tumors that exhibited high levels of HLA-G transcription by immunofluorescence of frozen sections and Western blot analysis of both tumor and healthy skin biopsies, using anti-HLA-G-specific monoclonal antibodies. This work provides evidence that HLA-G gene transcription and protein expression can be up-regulated ex vivo in melanoma. Our finding that several of the tumors studied expressed high levels of HLA-G provides additional clues as to how a tumor can be selected in vivo to escape from cytotoxic antitumor responses, constituting a new parameter to be considered in the design of therapeutic approaches aimed at enhancing antitumor immune responses.

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.

Introduction: Because of the potential hepatotoxicity of retinoids, prescription of isotretinoin is always very carefully made in healthy subjects, and prohibited in case of concomitant hepatopathy. Gilbert's syndrome consists of chronic, mild, unconjugated hyperbilirubinemia. In this syndrome, isotretinoin has been reported twice to be perfectly tolerated, and once even beneficial. We report here a new case of good tolerance and even improvement of a Gilbert's syndrome during isotretinoin therapy., Case Report: A 17-year-old man with Gilbert's syndrome presented with a nodulocystic acne. Topical agents had been inefficient, and cyclines bad tolerated. Thus isotretinoin has been gradually introduced, with a regular monitoring of the liver function. We observed a steady decrease of the bilirubinemia during the course of isotretinoin, and then a reappearance of hyperbilirubinemia as soon as posology was diminished and particularly after completion of isotretinoin therapy., Discussion: A review of the literature finds only very few cases of hepatic injuries caused by isotretinoin, contrary to etretinate. Safety of isotretinoin in Gilbert's syndrome was first observed in 1984, but its beneficial effects have only recently been described by Wang et al., and we report here a similar case. Pharmacological mechanisms remain hypothetic. However, considering the prevalence of Gilbert's syndrome and its usual first expression during postpubertal period, it seems to us interesting for therapeutic practice to know that isotretinoin is not less safe in these patients.

We report two cases of prolonged fever in deeply immunocompromised patients with AIDS who had been receiving trimethoprim-sulfamethoxazole (TMP-SMZ) as primary prophylaxis for several months. Investigations of the cause of fever yielded normal or negative findings except that the polymerase chain reaction (PCR) for Toxoplasma gondii in the blood was positive in both cases, and PCR of the bronchoalveolar lavage fluid was positive in one case. After a few days of treatment with pyrimethamine plus clindamycin, the two patients became afebrile and the T. gondii PCR became negative. The patients probably had disseminated toxoplasmosis attenuated by TMP-SMZ. PCR examination of blood for evidence of T. gondii genome may be useful in screening for causes of unexplained fever in patients with AIDS, even those who receive prophylaxis with TMP-SMZ.