[TeleSPOT Project : early detection of melanoma by teledermoscopy in general practice]. / Le projet TeleSPOT : Détection précoce du mélanome par télédermoscopie en Médecine générale.

Since decades the incidence of skin cancer is clearly rising. This alarming trend also applies to melanoma. It represents the 4th most common cancer in women and 6th in men in 2015. Early recognition and treatment reduce both morbidity and mortality. Screening is the cornerstone of secondary prevention. However, access to reliable and rapid diagnosis is hampered by several factors, including accessibility to specialized medicine. One of the solutions to this problem is to collaborate with the first-line medical care through a teledermatology system. The TeleSPOT project, Teledermoscopy Smartphone-based Pigmented lesion diagnosis Online Taskforce, aims to provide a remote diagnostic aid by dermatologists to distinguish suspect pigmented skin lesions and accelerate their management.

Depuis des décennies, l'incidence des cancers cutanés est en nette augmentation. Cette tendance alarmante s'applique également au mélanome. Il représente le 4ème cancer le plus fréquent chez la femme en 2015. Une prise en charge précoce permet de réduire la morbidité et la mortalité. Le dépistage représente la pierre angulaire de la prévention secondaire. Néanmoins, l'accès au diagnostic fiable et rapide est entravé par plusieurs facteurs limitants, notamment l'accessibilité à une Médecine spécialisée. Une des solutions à cette problématique est de collaborer avec la Médecine de première ligne par le biais de la télédermatologie. Le projet TeleSPOT, acronyme de Teledermoscopy Smartphone-based Pigmented lesion diagnosis Online Taskforce, a pour objectif de fournir une aide diagnostique à distance par des dermatologues afin de trier les lésions cutanées pigmentées suspectes et d'en accélérer la prise en charge.

Characterization of ß-cell plasticity mechanisms induced in mice by a transient source of exogenous insulin.

ß-Cell plasticity governs the adjustment of ß-cell mass and function to ensure normoglycemia. The study of how ß-cell mass is controlled and the identification of alternative sources of ß-cells are active fields of research. ß-Cell plasticity has been implicated in numerous physiological and pathological conditions. We developed a mice model in which we induced major ß-cell mass atrophy by implanting insulin pellets (IPI) for 7 or 10 days. The implants were then removed (IPR) to observe the timing and characteristics of ß-cell regeneration in parallel to changes in glycemia. Following IPR, the endocrine mass was reduced by 60% at day 7 and by 75% at day 10, and transient hyperglycemia was observed, which resolved within 1 wk. Five days after IPR, enhanced ß-cell proliferation and an increased frequency of small islets were observed in 7-day IPI mice. ß-Cell mass was fully restored after an additional 2 days. For the 10-day IPI group, ß-cell and endocrine mass were no longer significantly different from those of the control group at 2 wk post-IPR. Furthermore, real-time quantitative PCR analysis of endocrine structures isolated by laser capture microdissection indicated sequentially enhanced expression of the pancreatic transcription factors ß(2)/NeuroD and Pdx-1 post-IPR. Thus, our data suggest this mouse model of ß-cell plasticity not only relies on replication but also involves enhanced cell differentiation plasticity.

The Krüppel-like core promoter binding protein gene is primarily expressed in placenta during mouse development.

The human core promoter binding protein (hCPBP) has been identified as a DNA-binding protein involved in the regulation of TATA box-less genes like those encoding the pregnancy-specific glycoproteins. Structurally, hCPBP contains three zinc fingers in the C-terminal domain, which is highly conserved in a number of proteins that constitute the Krüppel-like family of transcription factors. In the present work, we report the molecular cloning of the mouse CPBP (mCPBP) and its expression pattern during development as well as in adult tissues. The mouse cDNA encodes a protein of 283 amino acids that share 94.4% of identity with the hCPBP. The highest level of mCPBP transcript was detected in placenta, and its expression was lower in total embryos and in adult tissues. We also show by in situ hybridization that during embryonic development the mCPBP gene is mainly expressed in extra-embryonic structures throughout gestation; essentially no specific expression was detected in embryonic tissues. Our data demonstrate that CPBP transcript is enriched in the trophoblastic tissue and strongly suggest that its encoded polypeptide regulates target genes involved in placental development and pregnancy maintenance.

OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6.

Transcription factors of the ONECUT class, whose prototype is hepatocyte nuclear factor (HNF)-6, are characterized by the presence of a single cut domain and by a peculiar homeodomain (Lannoy, V. J., Bürglin, T. R., Rousseau, G. G., and Lemaigre, F. P. (1998) J. Biol. Chem. 273, 13552-13562). We report here the identification and characterization of human OC-2, the second mammalian member of this class. The OC-2 gene is located on human chromosome 18. The distribution of OC-2 mRNA in humans is tissue-restricted, the strongest expression being detected in the liver and skin. The amino acid sequence of OC-2 contains several regions of high similarity to HNF-6. The recognition properties of OC-2 for binding sites present in regulatory regions of liver-expressed genes differ from, but overlap with, those of HNF-6. Like HNF-6, OC-2 stimulates transcription of the hnf-3beta gene in transient transfection experiments, suggesting that OC-2 participates in the network of transcription factors required for liver differentiation and metabolism.

Quantitative determination of several synthetic corticosteroids by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography.

A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.

Human TEF-5 is preferentially expressed in placenta and binds to multiple functional elements of the human chorionic somatomammotropin-B gene enhancer.

We report the cloning of a cDNA encoding the human transcription factor hTEF-5, containing the TEA/ATTS DNA binding domain and related to the TEF family of transcription factors. hTEF-5 is expressed in skeletal and cardiac muscle, but the strongest expression is observed in the placenta and in placenta-derived JEG-3 choriocarcinoma cells. In correlation with its placental expression, we show that hTEF-5 binds to several functional enhansons of the human chorionic somatomammotropin (hCS)-B gene enhancer. We define a novel functional element in this enhancer comprising tandemly repeated sites to which hTEF-5 binds cooperatively. In the corresponding region of the hCS-A enhancer, which is known to be inactive, this element is inactivated by a naturally occurring single base mutation that disrupts hTEF-5 binding. We further show that the binding of the previously described placental protein f/chorionic somatomammotropin enhancer factor-1 to TEF-binding sites is disrupted by monoclonal antibodies directed against the TEA domain and that this factor is a proteolytic degradation product of the TEF factors. These results strongly suggest that hTEF-5 regulates the activity of the hCS-B gene enhancer.

A one-nucleotide difference in a cAMP and phorbol ester response element leads to differential regulation of the human chorionic somatomammotropin A and B gene transcription.

Human chorionic somatomammotropin (hCS) is encoded by two highly related genes, hCS-A and hCS-B, located in the hGH/hCS gene locus. Both genes are expressed in the syncytiotrophoblast layer of the placenta and hCS release from trophoblast cells is known to be increased by cAMP and phorbol esters. However, it remains unclear whether this regulation acts at the level of hCS gene expression or secretion and whether both genes are affected. We examined the effects of cAMP and phorbol 12-myristate 13-acetate (PMA) on the transcription of the hCS-A and hCS-B genes. Transient expression experiments revealed a 7 bp cAMP- and PMA-responsive element (CRElhCS-A) spanning nucleotides-1102 to -1096 upstream of the hCS-A gene. In contrast, the homologous sequence upstream of hCS-B (CRElhCS-B), differing from CRElhCS-A by a single substitution, shows little or no response to cAMP. In band-shift assays, the CRElhCS-A oligonucleotide was shown to bind two factors related to CREBP and AP-1, whereas CRElhCS-B only competes for one of these complexes. Finally, Southwestern analysis revealed that the CRElhCS-A element binds two ubiquitous proteins of 100 kDa and 47 kDa respectively, whereas CRElhCS-B interacts only with the 47 kDa protein. Taken together, these results suggest that a 47 kDa protein binding to the CRElhCS-A and CRElhCS-B elements is involved in the PMA response of the hCS-A and hCS-B genes, and a 100 kDa protein plays a crucial role in cAMP regulation of the hCS-A gene.

A TEF-1 binding motif that interacts with a placental protein is important for the transcriptional activity of the hCS-B enhancer.

The transcriptional activity of the human placental lactogen genes (choriosomatomammotropic hormone, hCS) is controlled by tissue-specific enhancers located 4 kb downstream from their respective origins of transcription. The hCS-B enhancer is the strongest; its activity is mediated by synergism between two protein-binding sites (DF-3 and DF-4). The DF-4 site possesses a potential binding sequence for TEF-1, a known transcription factor. In this paper, we show by electrophoretic mobility-shift assays and antibody supershift experiments that TEF-1 does not bind to site DF-4. Mutations in the TEF-1-like binding motif of site DF-4 prevent formation of the DNA-protein complex, called complex f, in the presence of placental JEG-3 cell extracts. When HeLa cell extracts are used, another complex (complex c) is also affected. In transient expression experiments, TKCAT constructs linked to this mutated DF-4 site exhibit greatly reduced transcriptional activity when introduced into JEG-3 cells. Some cell lines contain both protein c and protein f (the proteins forming complexes c and f); when transfected, these lines display reduced DF-4-driven activity, suggesting that the two proteins could compete for the same DF-4 sequence. We conclude that protein f is important for the placenta-specific activity of the hCS-B enhancer. By UV cross-linking, we show that protein f is actually three polypeptides ranging in size from about 12 to 21 kD.

Efficient lipofection of human trophoblast cells in primary cultures.

Human choriosomatomammotropic hormone, also known as placental lactogen, is expressed in syncytiotrophoblast cells of the placenta. Studying transcriptional regulation of the genes coding for this hormone, we became interested in transfecting primary cultures of these trophoblast cells. In this study, we show that it is possible to transfect, by the lipofection method, these giant cells in an efficient and reproducible manner. We show the presence of an enhancer region downstream from the hCS-B gene, functionally active in these cells; furthermore, we demonstrate the placenta-specific characteristic of this enhancer, previously identified in a human choriocarcinoma cell line.

The transcriptional regulation of the growth hormone gene is conserved in vertebrate evolution.

Growth hormone (GH) gene expression in mammals is regulated by the interaction of the transcription factor Pit-1 with two binding sites within the proximal promoter. Four sequences, homologous to the mammalian Pit-1 motif occur in the rainbow trout (Oncorhynchus mykiss) GHII (rtGH) gene promoter, two of which partly overlap. The three regions containing these putative Pit-1 binding sequences were protected from deoxyribonuclease I digestion by nuclear extracts of GC cells, a rat pituitary tumor cell line producing Pit-1. In gel shift assays, nuclear proteins from GC cells and from trout pituitaries were found to interact specifically with one of these protected sites. Transfection experiments showed that the rtGH promoter is transcriptionally active in GC cells, the response being strongly enhanced in the presence of a cAMP analogue. The results demonstrate that rat Pit-1 binds to and activates the rtGH promoter, indicating that the basic mechanisms regulating GH gene transcription have been conserved between fish and mammals.

Detection of a retrovirus-related glycoprotein in immune complexes from patients with hematopoietic disorders.

Human sera from normal and leukemic individuals were found to contain various amounts of an antigen with determinants related to p15E and reverse transcriptase of retroviruses. Using a specific monoclonal antibody and the immuno-affinity purified antigen, we surveyed a series of sera and plasmas from normal individuals and hematological patients by competition radioimmunoassays and binding assays directed against this specific protein. It was detected in all the samples at various levels. The level of this 74-kd glycoprotein appeared to be related to a stimulation of the hematopoietic system. No free antibodies were found that could recognized the labelled purified antigen. Only immune complexes prepared from the blood of some hematological patients contained the specific antigen, but complexes prepared from the blood of normal individuals did not.

Bovine leukemia virus-related antigens in lymphocyte cultures infected with AIDS-associated viruses.

An earlier finding that lymphocytes from African patients with the acquired immune deficiency syndrome (AIDS) react with rabbit antiserum to purified antigens of bovine leukemia virus (BLV) prompted a study of the possible cross-reactions between a BLV-infected ovine cell line and human lymphocytes inoculated with a strain of lymphadenopathy syndrome-associated virus (LAV). A solid-phase radioimmunoassay was used to detect antigenic markers of the retroviruses. Crude extracts from short-term cultures of lymphocytes infected with LAV bound rabbit antisera to the LAV glycoprotein gp13 (molecular weight 13,000) and the BLV proteins p24 and gp51, but did not bind antibodies to the p24 of human T-cell leukemia virus type I (HTLV-I). Antiserum to LAV gp13 reacted with an ovine cell line producing BLV but also weakly with virus-free ovine cells. Lymphocyte cultures from four African patients with AIDS expressed BLV-related antigens within 6 to 10 days of culture, at the moment when particle-bound reverse transcriptase was produced. BLV-related antigens were induced in lymphocyte cultures from healthy individuals by addition of filtered supernatant or irradiated cells of the original culture. The antisera to BLV used in this study may prove useful for the detection of AIDS-associated viruses in short-term cultures of lymphocytes from AIDS patients or their contacts.

Production of monoclonal antibodies against HTLV-I proteins recognizing surface epitopes of live infected cells.

The proteins of HTLV-I virus, the only human retrovirus implicated in the etiopathogenesis of the T-cell leukemia, were previously studied with the use of monoclonal antibodies. Different groups have produced specific monoclonal antibodies that recognized the core proteins of the virus p19 and p24 and in one case a monoclonal specific of a gp21 protein. All these antibodies were shown to react with the virus-producing fixed cells. We also developed a battery of antibodies against p24 and p19 antigens from HTLV-I virus but the anti-p19 monoclonal antibodies happened to recognize epitopes exposed on the surface of live HTLV-I infected cells. One of the monoclonal antibody that bound to the surface of HTLV infected cells recognized a protein of an approximate mol. wt of 33 kilodalton (KD). These antibodies that bound to the live cells should be precious tools to study leukemic patients with T-cell leukemia and the evolution of the live cell populations during the course of the disease.

Study of the expression of a glycoprotein of retroviral origin in the plasma of patients with hematologic disorders and in the plasma of normal individuals.

A 74,000 molecular weight glycoprotein was purified from the plasma of a patient with chronic myelogenous leukemia in blast crisis. Monoclonal antibodies were produced in the mouse and used to characterize this protein. It was shown to contain p15E antigenic determinants and portions of a reverse transcriptase. The level of this protein was found to be elevated in leukemic patients with high white blood cell counts and also in some patients with other hematopoietic disorders as compared to the level measured in normal individuals. The level of the protein was strongly reduced in acute leukemia patients after intense chemotherapy treatment. We tentatively conclude that this protein is of endogenous retroviral origin and perhaps regulates hematopoietic tissues.

Expression of type C viral glycoproteins on P815 cells: higher expression of Mr 70,000 glycoprotein-containing glycoprotein on immunogenic variants.

Cells from P815 mastocytoma, a methylcholanthrene-induced tumor, were investigated for the presence of type C viral proteins on their surface. Little or no precipitation of purified Rauscher murine leukemia virus Mr 70,000 glycoprotein was observed with sera of mice immunized with P815 cells. However, a significant precipitation was obtained with sera from syngeneic or allogeneic mice immunized with P815 tumor cell variants obtained by mutagenesis. This difference was not detected when the sera were tested for precipitation of a pure Rauscher murine leukemia virus Mr 30,000 protein. The positive sera also precipitated a Mr 90,000 glycoprotein present on the membrane of all P815 cells. This glycoprotein was shown to contain endogenous ecotropic murine leukemia virus Mr 70,000 glycoprotein. On the membrane, a separate glycoprotein with a molecular weight of 90,000 containing gag gene products was also detected with a panel of reference antibodies. The level of glycoprotein containing Mr 70,000 glycoprotein on the cell membrane of the mutagenized cell variants was higher compared to the original P815 cells. The level of expression of the glycosylated gag precursor was the same on the original P815 cells and on the mutagenized cell variants.