Prediction of survival with second-line therapy in biliary tract cancer: Actualisation of the AGEO CT2BIL cohort and European multicentre validations

BACKGROUND: The benefit of second-line chemotherapy (L2) over standard first-line (L1) gemcitabine plus cisplatin (GEMCIS) or oxaliplatin (GEMOX) chemotherapy in advanced biliary tract cancer (aBTC) is unclear. Our aim was to identify and validate prognostic factors for overall survival (OS) with L2 in aBTC to guide clinical decisions in this setting. METHODS: We performed a retrospective analysis of four prospective patient cohorts: a development cohort (28 French centres) and three validation cohorts from Italy, UK and France. All consecutive patients with aBTC receiving L2 after GEMCIS/GEMOX L1 between 2003 and 2016 were included. The association of clinicobiological data with OS was investigated in univariate and multivariate Cox analyses. A simple score was derived from the multivariate model. RESULTS: The development cohort included 405 patients treated with L1 GEMOX (91%) or GEMCIS. Of them, 55.3% were men, and median age was 64.8 years. Prior surgical resection was observed in 26.7%, and 94.8% had metastatic disease. Performance status (PS) was 0, 1 and 2 in 17.8%, 52.4% and 29.7%, respectively. Among 22 clinical parameters, eight were associated with OS in univariate analysis. In multivariate analysis, four were independent prognostic factors (p < 0.05): PS, reason for L1 discontinuation, prior resection of primary tumour and peritoneal carcinomatosis. The model had the Harrell's concordance index of 0.655, a good calibration and was validated in the three external cohorts (N = 392). CONCLUSION: We validated previously reported predictive factors of OS with L2 and identified peritoneal carcinomatosis as a new pejorative factor in nearly 800 patients. Our model and score may be useful in daily practice and for future clinical trial design

Etude des interactions lympho-épithéliales et de leur modulation comme stratégie thérapeutique au cours de la maladie de Crohn

INTRODUCTION: La physiopathologie des maladies inflammatoires chroniques de l'intestin (MICI) fait intervenir à divers degrés des facteurs environnementaux, une prédisposition génétique et des facteurs microbiens qui interagissent avec un système immunitaire dysrégulé, entraînant des réponses aberrantes responsables d'une inflammation intestinale chronique. Le grand nombre de lymphocytes T présents dans la muqueuse iléale, leur importante diversité et leur interrelation avec le reste du système immunitaire, le microbiote et la barrière intestinale en font des acteurs majeurs de l'homéostasie intestinale avec un rôle certain dans la physiopathologie des MICI. Cependant, leurs fonctions, bien que partiellement découvertes, restent largement inconnues. OBJECTIF: L'objectif était donc d'étudier la muqueuse intestinale et en particulier les lymphocytes T et l'épithélium afin de préciser le rôle de ces sous-populations au cours des MICI. Ce travail s'est particulièrement intéressé à deux voies impliquées dans les interactions lympho-épithéliales : CD103 et NKG2D. MÉTHODES: Pour atteindre cet objectif, nous avons réalisé trois études consécutives : une analyse histologique des marges iléales provenant de pièces de résections iléo-coliques de patients subissant une résection iléale pour le traitement de la MC. Une étude phénotypique des lymphocytes de la muqueuse de patients atteints de MICI active inclus dans deux cohortes indépendantes : la cohorte REMIND et une cohorte de patients atteints de maladie active chez qui un traitement par biothérapie a été initié (cohorte ELYP). Nous avons ensuite développé un modèle de co-culture ex vivo entre cellules T de la muqueuse et organoïdes autologues pour étudier les interactions lympho-épithéliales et leur impact chez les patients atteints de MICI. Après le développement de ce modèle, nous avons étudié la modulation des 2 voies d'intérêt présentées (CD103 et NKG2D) et leur pertinence comme cibles thérapeutiques potentielles. RÉSULTATS: L'étude histologique des marges de résection iléale de patients opérés de MC iléale a mis en évidence que les lésions transmurales sont associées à une récidive postopératoire avec un impact potentiel sur la prise en charge des patients. Ce travail a également mis en évidence l'intérêt d'une étude approfondie des lymphocytes de la muqueuse. Via l'analyse phénotypique de deux cohortes indépendantes, nous avons trouvé des différences significatives dans la distribution des sous-types de cellules T de la muqueuse et mis en évidence l'importance de l'association des récepteurs CD103 et NKG2D à l'inflammation au cours des MICI. Nous avons démontré que les cellules T de la muqueuse de patients atteints de MC ont un potentiel cytotoxique accru dans des conditions allogéniques par rapport aux cellules T muqueuses de témoins. Le développement d'un modèle de co-culture entre des lymphocytes de la muqueuse et des organoïdes générés dans des conditions autologues nous a permis de démontrer une augmentation de la mort par apoptose des cellules épithéliales de patients atteints de MC dans ce modèle. Cet effet a été potentialisé par l'ajout de LPS, censé mimer, de façon simplifiée, les produits bactériens potentiellement présents in vivo dans l'intestin. L'utilisation d'anticorps bloquant les voies d'intérêt CD103 et NKG2D a permis l'inhibition de cet effet confirmant l'intérêt thérapeutique potentiel de ces voies. CONCLUSION: Nous avons ainsi pu démontrer que les interactions lympho-épithéliales jouent un rôle majeur dans la physiopathologie des MICI et plus particulièrement de la MC, avec un rôle délétère de certaines populations immunitaires dont les cellules T exprimant CD103 et NKG2D. Ce travail ouvre la voie à de multiples perspectives, notamment grâce à la complexification de ce modèle de co-culture autologue qui permettra dans un futur proche de récapituler ex vivo toute la complexité de la muqueuse intestinale.IINTRODUCTION: The pathophysiology of Inflammatory Bowel Diseases (IBD) involves in various degrees environmental triggers, genetic predisposition and microbial factors interacting with a dysregulated immune system resulting in aberrant responses responsible for chronic intestinal inflammation. The large number of T lymphocytes present in the ileal mucosa, their important diversity and their interrelation with the rest of the immune system, the microbiota and the intestinal barrier make them major players in intestinal homeostasis with a definite role in the pathophysiology of IBD. However, their functions, although partially discovered, remain largely unknown. OBJECTIVE: The objective was therefore to study the intestinal mucosa and in particular the T lymphocytes and the epithelium in order to clarify the role of these subpopulations during IBD. This work particularly focused on two pathways involved in lympho-epithelial interactions: CD103 and NKG2D. METHODS: To achieve this objective we performed three consecutive studies: a histological analysis of ileal margins from ileocolic resections specimens of patients undergoing ileal resection for the treatment of Crohn's Disease (CD). A phenotypic study of the mucosal lymphocytes of patients with active IBD included in two independent cohorts: the REMIND cohort and a cohort of patients with active disease in whom biotherapy treatment was initiated (ELYP cohort). We then developed an autologous organoid-mucosal T cell ex vivo co-culture model to study lympho-epithelial interactions and their impact in IBD patients. After the development of this model, the modulation of the 2 pathways of interest presented (CD103 and NKG2D) was studied and their relevance as potential therapeutic targets. RESULTS: The histological study of ileal resection margins of operated ileal CD patients highlighted that transmural lesions are associated with postoperative recurrence with a potential impact on patient management. This work also highlighted the value of extensive study of mucosal lymphocytes. Using phenotypic analysis of two independent cohorts, we found significant differences in the distribution of mucosal T cell subtypes and highlighted the importance of the CD103 and NKG2D receptors association in inflammation during IBD. We demonstrated that mucosal T cells from CD patients have an increased cytotoxic potential under allogeneic conditions compared to mucosal T cells from control patients. The development of a co-culture model between mucosal lymphocytes and organoids generated under autologous conditions allowed us to demonstrate an increase in apoptotic death of epithelial cells from CD patients in this model. This effect was potentiated by the addition of LPS, which is supposed to mimic, in a simplified way, the bacterial products potentially found in vivo in the intestine. The use of antibodies blocking the CD103 and NKG2D pathways of interest inhibited this effect by apparently different mechanisms. CONCLUSION: Through the access to a large panel of human data and specimens from IBD patients in established cohorts, we were able to demonstrate step by step that lympho-epithelial interactions play a major role in the pathophysiology of IBD and more specifically Crohn's disease, with a deleterious role for certain immune cell populations in inflammation including T cells expressing CD103 and NKG2D. This work paves the way to multiple perspectives, in particular thanks to the complexification of this autologous co-culture model which will make it possible in the near future to recapitulate ex vivo the entire complexity of the intestinal mucosa.

Contributions to the study of the natural history of Mycobacterium ulcerans

Mycobacterium ulcerans cause l'ulcère de Buruli, qui est une maladie tropicale négligée signalée dans 36 pays. M. ulcerans est étroitement apparenté à Mycobacterium marinum (M. marinum). Il sécrète des toxines mycolactones qui sont les principaux facteurs de virulence de la bactérie, responsable des ulcères sur la peau et les tissus sous-jacents et les os. Le réservoir et le mode de contamination par M. ulcerans ne sont pas formellement connus, ce qui empêche l'établissement d'une prophylaxie raisonnée. Dans une perspective "One Health", mon travail de thèse contribue à démasquer le(s) réservoir(s) et le(s) mode(s) de contamination par M. ulcerans.Ma revue de la littérature met en évidence la taxonomie de ces mycobactéries productrices de mycolactone et de M. marinum afin de comprendre l'épidémiologie de l'ulcère de Buruli. Mes travaux expérimentaux ont conduit au développement de milieux de décontamination et de culture pour M. ulcerans ; à la révélation d'une nouvelle chaîne de contamination par M. ulcerans suite à la préparation de viande de brousse contaminée, et à la démonstration du rôle de la toxine mycolatone dans la niche environnementale de M. ulcerans.Mycobacterium ulcerans, causes Buruli Ulcer which is a neglected tropical disease reported in 36 countries. M. ulcerans is closely related to Mycobacterium marinum (M. marinum). It secretes mycolactone toxins which are the main virulence factors of the bacterium, responsible for ulcers on skin and underlying tissues and bones. The reservoir and mode of contamination by M. ulcerans are not formally known, hindering the establishment of a reasoned prophylaxis. From a "One Health" perspective, my thesis work contributes to unmasking the reservoir(s) and mode(s) of contamination by M. ulcerans.My review of the literature highlights the taxonomy of these mycolactone-producing mycobacteria and M. marinum in order to understand the epidemiology of Buruli Ulcer. My experimental work has led to the development of decontamination and culture media for M. ulcerans; the revelation of a new chain of contamination by M. ulcerans following the preparation of contaminated bushmeat , and the demonstration of the role of the mycolatone toxin in the environmental niche of M. ulcerans

Reply to: Inflammatory bowel disease with peritoneal metastases: A complex and extremely variable disease

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Temperature affects the biology of Schmidtea mediterranea

International audienceStudies of tissue regeneration and host-pathogen interactions using the model planarian Schmidtea mediterranea have been performed at an experimental temperature of 19 degrees C. S. mediterranea planarians exposed to 19 degrees C-32 degrees C were observed for survival, mobility, feeding and regeneration for three months and elimination of the Staphylococcus aureus pathogen over six days. S. mediterranea planarians died at 30 degrees C-32 degrees C after 18 days of observation but tolerated temperatures of 19 degrees C up to 28 degrees C with non-significant differences in mobility and feeding behavior. Genetic malleability tested by RNAi feeding was still efficient at 26 degrees C and 28 degrees C. Concerning the immune capacity of planarians, we reported an exacerbation of the immune response in worms infected by S. aureus at 26 degrees C and 28 degrees C. These observations suggest a temperature modulation of planarian stem cells and illustrate the importance of modulating experimental temperature when using planarians as model organisms to study regeneration and immune response

Screening anti-infectious molecules against Mycobacterium ulcerans: A step towards decontaminating environmental specimens

International audienceMycobacterium ulcerans, a non-tuberculous mycobacterium responsible for Buruli ulcer, resides in poorly defined environmental niches in the vicinity of stagnant water. Very few isolates have been confirmed. With a view to culturingM.ulceransfrom such contaminated environmental specimens, we tested thein vitrosusceptibility of theM.ulceransCU001 strain co-cultivated with XTC cells to anti-infectious molecules registered in the French pharmacopoeia. We used a standardised concentration to identify molecules that were inactive againstM.ulceransand which could be incorporated into a decontaminating solution. Of 116 tested molecules, 64 (55.1%) molecules were ineffective against M. ulcerans CU001. These included 34 (29.3%) antibiotics, 14 (12%) antivirals, eight (6.8%) antiparasitics, and eight (6.8%) antifungals. This left 52 molecules which were active againstM.ulceransCU001. Three of the inactive antimicrobial molecules (oxytetracycline, polymyxin E and voriconazole) were then selected to prepare a decontamination solution which was shown to respectM.ulceransCU001 viability. These three antimicrobials could be incorporated into a decontamination solution to potentially isolate and cultureM.ulceransfrom environmental samples

Translocating Mycobacterium ulcerans: An experimental model

Mycobacterium ulcerans is a non-tuberculous environmental mycobacterium responsible for extensive cutaneous and subcutaneous ulcers in mammals, known as Buruli ulcer in humans. M. ulcerans has seldom been detected in the faeces of mammals and has not been detected in human faeces. Nevertheless, the detection and isolation of M. ulcerans in animal faeces does not fit with the current epidemiological schemes for the disease. Here, using an experimental model in which rats were fed with 10(9) colony-forming units of M. ulcerans, we detected M. ulcerans DNA in the faeces of challenged rats for two weeks and along their digestive tract for 10 days. M. ulcerans DNA was further detected in the lymphatic system including in the cervical and axillary lymph nodes and the spleen, but not in any other tissue including healthy and broken skin, 10 days post-challenge. These observations indicate that in some herbivorous mammals, M. ulcerans contamination by the digestive route may precede translocation and limited contamination of the lymphatic tissues without systemic infection. These herbivorous mammals may be sources of M. ulcerans for exposed populations but are unlikely to be reservoirs for the pathogen

Mycobacterium ulcerans Experimental Dormancy

International audienceWhether Mycobacterium ulcerans , the etiological agent of Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, has not been established. Six M. ulcerans strains were exposed to a progressive depletion in oxygen for 2 months, using the Wayne model of dormancy previously described for M. tuberculosis , and further examined by microscopy after staining of dynamic, dormant, and dead mycobacteria (DDD staining), microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls. Mycobacterium ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile red–stained intracellular lipid droplets and a 14- to 20-day regrowth when exposed to ambient air, consistent with dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD staining and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans , warrants further investigations in the natural ecological niches where M. ulcerans thrive as well as in Buruli ulcer lesions

Confirming Autochthonous Buruli Ulcer Cases in Burkina Faso, West Africa

International audienceEnvironmental Mycobacterium ulcerans causes a disabling skin disease called Buruli ulcer. Recent studies completed the knowledge of the evolving geographic extension and epidemiology of Buruli ulcer in West Africa, where Côte d’Ivoire is reporting the highest number of cases. We report seven polymerase chain reaction-documented patients in Burkina Faso, a neighboring country of Côte d’Ivoire, where previously Buruli ulcer cases were confirmed primarily using clinical arguments

Disseminated Mycobacterium ulcerans Infection in Wild Grasscutters (Thryonomys swinderianus), Côte d’Ivoire

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