A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BNm for “BN mutated.” In BNm rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4+ CD25bright regulatory T cells (Treg) is defective in BNm rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BNm rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BNm rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BNm×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to <i>FAM111B </i>mutations

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder

L'apoptose dans l'épithélium intestinal humain (hiérarchisation des étapes d'exécution - contrôle par le monoxyde d'azote (NO))

En physiologie intestinale, l'apoptose est une fonction qui, en assurant une mort cellulaire dite "propre", participe au maintien de l'homéostasie de la barrière épithéliale...L'objectif de cette thèse était double. D'une part, ce travail visait à déterminer 1) l'expression dans l'épithélium intestinal humain de la protéine DFF45, impliquée dans la dégradation internucléosomale de l'ADN dans le système murin, et 2) son éventuel rôle dans l'apoptose épithéliale. D'autre part, ce travail visait à explorer le rôle pro-apoptotique d'un médiateur physiologique, le monoxyde d'azote (NO), vis-à-vis de cellules épithéliales intestinales en culture, et à décrypter les voies de signalisation impliquées. Nos résultats montrent que 1) l'épithélium colique humain normal exprime DFF45 et 2) l'expression de DFF45 est dépendante de la phase de croissance, dans la lignée cancéreuse colique humaine HT29-Cl16E.....In intestinal physiology, apoptosis is a cellular function leading to a "clean" cell death that participates in epithelial barrier homeostasis. As any other physiological function, apoptosis comprises execution mechanisms which are coupled to intracellular signaling systems and cellular receptors, and are activated by extracellular mediators. In colonic cancer, a deficient expression of apoptosis-activating receptors (Fas) or receptor-proximal proteins could account for the resistance of various colonic cancer cell lines to apoptotic stimuli. The execution mechanisms of apoptosis in both human normal intestinal epithelium and colonic cancers have not been extensively elucidated yet. Finally, the capacity of some physiological mediators to regulate apoptosis in the human normal intestinal epithelium or in colonic cancers is not well known. The aims of this work were twofold. The first aim was to examine the expression by the human intestinal epithelium of DFF45, a protein known to be involved in internucleosomal DNA fragmentation in mice, and to determine its role in the apoptosis of the intestinal barrier.....NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Contrôle métabolique de la réponse des cellules épithéliales au stress oxydatif (une nouvelle approche par manipulation de la source hydrocarbonée des cellules en culture)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Implication de deux effecteurs moléculaires ubiquitaires, calréticuline et ADAM15, dans la morphogenèse et la différenciation des néoplasies coliques (une approche fondée sur des biocollections)

L'oncogenèse colique fait appel à la notion de morphogenèse subordonnée à des anomalies génétiques spécifiques agissant par l'intermédiaire d'effecteurs moléculaires, dont les fonctions peuvent être prédites par des modèles animaux. Cependant, au cours de la progression tumorale, la diversification des anomalies génétiques et des effecteurs se prête difficilement à la modélisation animale. L'exploitation de biocollections tumorales humaines est alors la clef de la modélisation du rôle des effecteurs potentiels. Ce travail de thèse, fondé sur l'utilisation de biocollections annotées de cancers colorectaux (CCR), vise à 1) examiner dans les CCR l'expression in situ de la calréticuline et de l'ADAM15, 2 protéines impliquées dans l'adhésion, la migration et la différenciation épithéliale et 2) corréler leur expression avec les données génétiques, clinicopathologiques et pronostiques des CCR. Les points importants sont : - une diminution d'expression de la calréticuline et de son récepteur CD91 dans les cellules néoplasiques des CCR, par rapport à l'épithélium colique normal apparié. Le maintien de l'expression de la calréticuline dans les CCR est associée à la différenciation mucineuse, en accord avec son rôle de chaperonne de la mucine MUC2 (article 1). - une perte d'expression d'ADAM15 dans les CCR dé-différenciés, associée à la progression tumorale via un mécanisme de transition épithélio-mésenchymateuse, et à un pronostic péjoratif (article 2). Cette étude permet de proposer une hiérarchisation spatiale et temporelle des effecteurs moléculaires dans les phénomènes de subordination de la progression néoplasique colique humaine.Colonic oncogenesis is linked to the concept of morphogenesis, subordinated to specific genetic abnormalities involving molecular effectors whose functions can be predicted by animal models. However, during tumor progression, the diversity of genetic abnormalities and effectors can hardly be modelized in animal models. The exploitation of human tumor tissue collections is pivotal in the modelization of potential effectors functions. The aims of this work, based on biocollections of colorectal cancers (CRC) with clinical annotations, were 1) to examine the in situ expression of calreticulin and ADAM15, 2 proteins involved in cell adhesion, migration and epithelial differentiation, and 2) to correlate their expression with the genetic, clinicopathological and prognostical data of CRC. The main points of this study are: - a down-regulation of calreticulin expression and its receptor CD91 in neoplastic cells of CRC, compared with normal paired epithelium. Maintenance of calreticulin expression is associated with mucinous differentiation, in line with its role as a chaperone of the mucin MUC2 (article 1). - a loss of ADAM15 expression in de-differentiated CRC, associated with tumor progression through an epithelial-to-mesenchymal transition, and with a poor prognosis (article 2). This study allows to propose a spatial and temporal ordering of molecular effectors in the subordinated events occurring in colonic neoplasia progression.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF