Clinical proteomics : a focus on transformed human T and B lymphocytes

Résumé large public La protéomique clinique est une discipline qui vise l'étude des protéines dans un but diagnostique ou thérapeutique. Nous avons utilisé cette approche pour étudier les lymphocytes T «tueurs » ou cytotoxiques qui font partie des globules blancs du système sanguin et agissent dans la lutte contre les infections et les tumeurs. Ces cellules sont impliquées dans l'immunothérapie cellulaire qui se fonde sur la capacité naturelle des ces lymphocytes à repérer les cellules tumorales et à les détruire. L'introduction du gène de la télomérase dans les lymphocytes T résulte en une prolongation de leur longévité, ce qui en ferait des candidats intéressants pour l'immunothérapie cellulaire. Il subsiste cependant des doutes quant aux conséquences de l'utilisation de ces lymphocytes «immortalisés ». Pour répondre à cette question, nous avons comparé le profile protéique de lymphocytes T cytotoxiques «jeunes » et vieux » avec celui des lymphocytes «immortalisés ». Nous avons trouvé que ces derniers présentent une double face et partagent à la fois les caractéristiques de la jeunesse et de la vieillesse. Dans une seconde étude de protéomique clinique, nous nous sommes penchés sur les lymphocytes B «immortalisés » cette fois-ci non pas avec la télomérase, mais avec le virus d'Epstein-Barr. Ces derniers sont utilisés comme modèle dans l'étude de la leucodystrophie, une maladie génétique rare qui affecte le cerveau. Notre but est d'identifier des marqueurs biologiques potentiels qui pourraient aider le diagnostic et le traitement de cette maladie neurodégénérative. Nous avons pour ce faire comparé les profiles protéiques des lymphocytes B «immortalisés » provenant d'individus sains et malades. Malheureusement, notre analyse n'a pas révélé de différences notoires entre ces deux classes de lymphocytes. Ceci nous permet toutefois de conclure que la maladie n'affecte pas la synthèse des protéines de manière prépondérante dans ces cellules sanguines. En résumé, le travail présenté dans cette thèse montre à la fois le potentiel et les limites de l'analyse des protéines lymphocytaires, dans différentes situations biologiques. Résumé La protéomique clinique ouvre la porte vers de multiples horizons relatifs au traitement de diverses maladies. Ce domaine particulier alliant la protéomique à la médecine, implique l'intervention de la biologie moléculaire et cellulaire. Dans notre étude, nous nous sommes d'abord intéressés aux lymphocytes T CD8+ cytotoxiques dans le contexte de l'immunothérapie adoptive. Le fondement de cette thérapie repose sur la capacité naturelle de ces lymphocytes à reconnaître les cellules tumorales et à les détruire chez les patients atteints de cancer. L'introduction du gène de la transcriptase réverse de la télomérase (hTERT) dans les lymphocytes T humains permet de rallonger leur durée de vie, sans toutefois induire d'altérations liées à la transformation. Cependant, des incertitudes subsistent quant à la ressemblance physiologique et biochimique entre ces cellules surexprirnant la télomérase et les cellules normales. Afin de répondre à cette question, nous avons comparé l'expression des protéines de lymphocytes humains T CD8+ «jeunes » et «vieux »avec celle de lymphocytes transduits avec hTERT. Nous avons trouvé que les lymphocytes T surexprimant la télomérase ont un profile protéique intermédiaire, avec certaines expressions protéiques similaires aux jeunes cellules T et d'autres se rapprochant des cellules vieilles. Dans la seconde partie de notre étude, nous nous sommes intéressés aux lymphocytes B transformés avec le virus d'Epstein-Barr provenant de patients atteints d'une maladie génétique rare du cerveau, la leucodystrophie. Dans cette maladie, des mutations dans le facteur de transcription eIF2B, impliqué dans la synthèse protéique, ont été trouvées. Afin d'analyser les conséquences de ces mutations et de trouver des biomarqueurs spécifiques à cette maladie, nous avons effectué une analyse protéomique des lymphoblastes provenant de malades et d'individus sains. Nous avons trouvé que les mutations dans le complexe ubiquitaire eIF2B n'affectent pas de manière significative l'expression des protéines des lymphoblastes mutés. En conclusion, notre travail illustre le potentiel et les limitations des technologies protéomiques utilisées pour disséquer l'implication des protéines dans différentes situations biologiques. Summary Clinical proteomics opens the door to multiple applications related to the treatment of diseases. This particular field is at the crossroad of proteomics and medicine and involves tools from cellular and molecular biology. We focused first our investigations on cytotoxic T cells in the context of adoptive immunotherapy, which is an interesting and evolving field. The basis of this therapy relies on the natural capacity of cytotoxic CD8+ T lymphocytes in recognizing tumor cells and destroying them in cancer patients. As their number is reduced, the idea would be to use transformed T lymphocytes with extended life span. Overexpression of telomerase into human T lymphocytes results in the extension of their replicative life span, but it still remains unclear whether these cells are physiologically indistinguishable from normal ones. To address this question, we compared the proteome of young and aged CD8+ T lymphocytes with that of T cells transduced with hTERT and found that the latter cells displayed an intermediate protein pattern, sharing similar protein expression with young, but also with aged T cells. We were then interested in studying Epstein-Barr virus transformed B lymphocytes in the context of a rare human brain genetic disorder called leukodystrophy. In this disease, mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported. In order to analyze the functional consequences of the mutations and to find out specific biomarkers of eIF2B-related disorders, proteomic and peptidomic studies were carried out on lymphoblasts from eIF2Bmutated patients versus healthy patients. Following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not appear to significantly affect the proteome of the mutated lymphoblasts extracts. To conclude, our work emphasizes the potentials and the limitations of the proteomic technologies used to analyze the role of lymphocyte proteins in different biological situations.

Prevalence of subfoveal nodules in Coat's disease

Purpose:Coats' disease is a non-hereditary condition characterized by idiopathic retinal telangiectasia, and exudative retinopathy. Although the exudation often spreads from the main areas of telangiectasia, there is a preferential accumulation of exudation in the macular area in Coats' disease. A subfoveal nodule has usually been described in the context of resolution of macular exudates after treatment of peripheral retinal telangiectasis. Nevertheless, a recent reports stressed out an uncommon prominent subfoveal nodule with peripheral exudates as initial presentation of Coats'disease. The purpose of this study was to report the prevalence of this presentation in a cohort of patients. Methods:All consecutive patients with Coats' disease referred to the Jules Gonin Eye Hospital between January 1979 and July 2006 were included. All charts were screened for a clear cut subfoveal circular lesion on fundus photographies at initial presentation. Results:95 patients suffering of Coat's disease were enrolled. 33 out of 95 patients had subtotal or total exudative retinal detachment, which impeded macular examination. 14 out of 62 (22.6%) resting patients presented with a clear cut prominent circular subfoveal lesion at initial presentation. All patients had unilateral disease. Mean age was 5.6 ± 3.5 year old at initial presentation. There were 4 females and 10 males. Pigmentation and size of the nodule were not homogenous. Mean diameter was 1.1 ± 0.5 optic disc diameter. Conclusions:The present study shows that subfoveal nodule is not such a rare primary presentation of Coats' disease in contrast to what it has been previously reported in the literature. Thus the initial finding of prominent subfoveal nodule associated with peripheral retinal findings made the diagnosis of Coats' disease highly likely.Physicians should be aware that a proeminent subfoveal nodule is a common initial presentation of Coats' disease as it can be confused clinically with Retinoblastoma.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

Lack of the alanine-serine-cysteine transporter 1 causes tremors, seizures, and early postnatal death in mice.

The Na(+)-independent alanine-serine-cysteine transporter 1 (Asc-1) is exclusively expressed in neuronal structures throughout the central nervous system (CNS). Asc-1 transports small neutral amino acids with high affinity especially for D-serine and glycine (K(i): 8-12 microM), two endogenous glutamate co-agonists that activate N-methyl-D-aspartate (NMDA) receptors through interacting with the strychnine-insensitive glycine binding-site. By regulating D-serine (and possibly glycine) levels in the synaptic cleft, Asc-1 may play an important role in controlling neuronal excitability. We generated asc-1 gene knockout (asc-1(-/-)) mice to test this hypothesis. Behavioral phenotyping combined with electroencephalogram (EEG) recordings revealed that asc-1(-/-) mice developed tremors, ataxia, and seizures that resulted in early postnatal death. Both tremors and seizures were reduced by the NMDA receptor antagonist MK-801. Extracellular recordings from asc-1(-/-) brain slices indicated that the spontaneous seizure activity did not originate in the hippocampus, although, in this region, a relative increase in evoked synaptic responses was observed under nominal Mg(2+)-free conditions. Taken together with the known neurochemistry and neuronal distribution of the Asc-1 transporter, these results indicate that the mechanism underlying the behavioral hyperexcitability in mutant mice is likely due to overactivation of NMDA receptors, presumably resulting from elevated extracellular D-serine. Our study provides the first evidence to support the notion that Asc-1 transporter plays a critical role in regulating neuronal excitability, and indicate that the transporter is vital for normal CNS function and essential to postnatal survival of mice.

N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Phenotypic and molecular characterization of the onset of neuropathy in rodent models of diabetes mellitus

Abstract : The principal focus of this work was to study the molecular changes leading to the development of diabetic peripheral neuropathy (DPN). DPN is the most common complication associated with both type I and II diabetes mellitus (DM). This pathology is the leading cause of non-traumatic amputations. Even though the pathological and morphological changes underlying DPN are relatively well described, the implicated molecular mechanisms remain poorly understood. The following two approaches were developed to study the development of DPN in a rodent model of DM type I. As a first approach, we studied the implication of lipid metabolism in DPN phenotype, concentrating on Sterol Response Element Binding Protein (SREBP)-lc which is the key regulator of storage lipid metabolism. We showed that SREBP-1c was expressed in peripheral nerves and that its expression profile followed the expression of genes involved in storage lipid metabolism. In addition, the expression of SREBP-1c in the endoneurium of peripheral nerves was dependant upon nutritional status and this expression was also perturbed in type I diabetes. In line with this, we showed that insulin elevated the expression of SREBP-1c in primary cultured Schwann cells by activating the SREBP-1c promoter. Taken together, these findings reveal that SREBP-1c expression in Schwann cells responds to metabolic stimuli including insulin and that this response is affected in type I diabetes mellitus. This suggests that disturbed SREBP-1c regulated lipid metabolism may contribute to the pathophysiology of DPN. As a second approach, we performed a comprehensive analysis of the molecular changes associated with DPN in the Akital~1~+ mouse which is a model of spontaneous early-onset type I diabetes mellitus. This mouse expresses a mutated non-functional isoform of insulin, leading to hypoinsulinemia and hyperglycaemia. To determine the onset of DPN, weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Akital+/+ mice during the first three months of life. A decrease in MNCV was evident akeady one week after the onset of hyperglycemia. To explore the molecular changes associated with the development of DPN in these mice, we performed gene expression profiling using sciatic nerve endoneurium and dorsal root ganglia (DRG) isolated from early diabetic male Akita+/+ mice and sex-matched littermate controls. No major transcriptional changes were detected either in the DRG or in the sciatic nerve endoneurium. This experiment indicates that the phenotypic changes observed during the development of DPN are not correlated with major transcriptional alterations, but mainly with alterations at the protein level. Résumé Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1 c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita+/+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique. Résumé : Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita~~Z~+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique.

CELLULAR AND MOLECULAR STUDY OF SUBCORTICAL BAND HETEROTOPIA IN THE MOUSE MUTANT HECO

The HeCo mouse model is characterized by a subcortical heterotopia formed by misplaced neurons normally migrating into the superficial cortical layers. The mutant mouse has a tendency to epileptic seizures. In my thesis project we discovered the mutated Eml1 gene, a member of the echinoderm microtubule-associated protein (EMAP) family, in HeCo as well as in a family of three children showing complex malformation of cortical development. This discovery formed an important step in exploring the pathogenic mechanisms underlying the HeCo phenotype. In vitro results showed that during cell division the EML1 protein is associated with the midbody and a mutated version of Eml1 highlighted an important role of the protein in the astral MT array during cell cycle. In vivo, we found that already at an early age of cortical development (E13), ectopic progenitors such as RGs (PAX6) and IPCs (TBR2) accumulate in the IZ along the entire neocortex. We demonstrated that in the VZ of the HeCo mouse, spindle orientation and cell cycle exit are perturbed. In later stages (E17), RG fibers are strongly disorganized with deep layer (TBR1) and upper layer (CUX1) neurons trapped within an ectopic mass. At P3, columns of upper layer neurons were present between the heterotopia and the developing cortex; these columns were also present at P7 but at lesser extent. Time lapse video recording (E15.5) revealed that the parameters characterizing the migration of individual neurons are not disturbed in HeCo; however, this analysis showed that the density of migrating neuron was smaller in HeCo. In conclusion, truncated EML1 is likely to play a prominent role during cell cycle but also acts on the cytoskeletal architecture altering the shape of RG fibers thus influencing the pattern of neuronal migration. The signal transduction between external cues and intracellular effector pathways through MTs may be secondary but sustains the heterotopia development and further studies are needed to clarify the impact of EML1 in progenitors versus post-mitotic cells.

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

Les biomarqueurs prédictifs dans le cancer colorectal [Predictive biomarkers in colorectal cancer]

While for many years the diagnosis and therapy of colon cancer did not change drastically, recently new drugs (irinotecan and oxaliplatin, used in adjuvant or neo-adjuvant approaches) and even more recently the introduction of therapies targeting the epidermal growth factor receptor (EGFR) through the monoclonal antibodies cetuximab and panitumumab, are revolutionizing the field. The finding that only patients with a tumor with a wild type (non mutated) KRAS gene respond to anti-EGFR therapy has also affected the way pathologists address colorectal cancer. Molecular analysis of the KRAS gene has become almost a routine in a very short period of time. Pathologists will have to be prepared for a new era: from standard morphology based diagnostic procedures to the prediction of response to therapy using molecular tools.

Molecular Analysis of the PTEN Gene in a Choroidal Schwannoma in the Context of a Hamartomatous Syndrome

Purpose: To investigate the molecular involvement of PTEN, a tumor suppressor gene, in a case of cellular pigmented choroidal Schwannoma in a patient with hamartomatous syndrome due to heterozygous PTEN germline mutation. Methods: Histopathological, immunohistochemical, and electron microscopy analyses were performed by standard procedures. Paraffin-embedded samples of normal and tumor eye tissues were collected and DNA was extracted. A 145 bp region flanking the heterozygous c.406T>C mutation in exon 5 of PTEN was amplified by PCR and sequenced. To evaluate the allelic status of PTEN in the tumor sample, we cloned different PCR products in E. coli using a TA cloning procedure. Results: Histopathology demonstrated a posterior choroidal mass measuring 1.3 x 1.6 x 1.4 cm. The tumor was composed by fascicles of spindle cells with wavy cytoplasm. No Verrocay bodies could be identified. Scattered histiocytes with clear cytoplasm were present. By immunohistochemistry, the cells were expressing S100 and focally Melan A proteins. Pericellular type IV collagen could be demonstrated. Interlacing cytoplasmic processes covered by thick basement membrane could be found by electron microscopy as well as few premelanosomes. Moderate PTEN expression by immunohistochemistry was identified in some cells. As expected, the germline mutation could be detected by DNA sequencing in both the paraffin-embedded normal and tumor eye tissues. Analysis of 33 E. coli colonies bearing clones from the tumor eye tissue DNA surprisingly revealed that most of them contained the PTEN wild-type allele (29 vs. 4, Fisher's test p-value = 0.002). Conclusions: This is the first reported case of choroidal cellular Schwannoma arising in the context of a PTEN hamartomatous syndrome. Allelic analysis of PTEN in the tumor suggests a statistically-significant partial loss of heterozygozity in favor of the wild-type allele. Our findings are in clear contrast with what is usually observed in cancer tissues, for which mutated alleles of tumor suppressor genes are usually brought to homozygosity. Similar results were previously reported in human non-Hodgkin's lymphomas, displaying an overexpression of the wild-type form of the tumor suppressor gene p53. We are in the process of investigating additional DNA derived from other fresh and paraffin-embedded tissues from the patient, in order to gain insights on the molecular bases of PTEN involvement in this rare choroidal Schwannoma.

Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under Accession Nos E-MTAB-990 and E-MTAB-1026. © 2013 Swiss Institute of Bioinformatics. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of machado-joseph disease.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.