A Guide for Ex Vivo Handling and storage of stool samples Intended for Fecal Microbiota transplantation

International audienceOwing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMt) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a-80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation

Impact of human gut microbiota in the progression of non-alcoholic fatty liver diseases (NAFLD)

Les hépatopathies métaboliques non liées à l'alcoolisme (NAFLD) regroupent un spectre de maladies hépatiques allant de la stéatose hépatique (NAFL), en passant par la fibrose (NASH), puis la cirrhose en l’absence de consommation d’alcool, d’infection virale ou d’autres causes identifiées, et parfois le développement d’un carcinome hépatocellulaire (CHC). Du fait de sa prévalence importante, allant jusqu’à un quart de la population générale européenne et de ses conséquences néfastes sur la santé des personnes qui en sont atteintes, les NAFLD représentent un problème de santé publique majeur.Cette maladie hépatique a été associée à des changements du microbiote intestinal (MI) et des voies métaboliques dans lesquelles ce microbiote est impliqué. Chez la souris, le MI contribue à l’exacerbation ou à la protection de cette maladie.L’objectif de la thèse est de déterminer si chez l’Homme, le MI contribue également à cette susceptibilité individuelle aux NAFLD observée chez la souris.Une procédure standardisée complète pour préparer, stocker et gérer les transplants fécaux humains a été développée et mise en place (Burz et al., 2019).Des souris exemptes d’organismes pathogènes spécifiques (EOPS), prétraitées avec un mélange d’antibiotiques, ont été colonisées avec les microbiotes dérivés d’un individu sain ou de patients à différents stades de la NAFLD. Ces souris ont ensuite été soumises à un régime riche en lipides et en fructose, afin d’explorer l’effet des microbiotes installés sur l’apparition et l’évolution de la NAFLD induite.Ce projet a notamment permis de démontrer à travers le transfert du microbiote fécale humain, et à consommation énergétique alimentaire constante, le transfert du surpoids. Le microbiote fécal humain NAFL, aggravant la stéatose hépatique chez la souris, mais atténuant l’inflammation hépatique et caecale.Cette thèse apporte un éclairage sur l'impact du microbiote intestinal dans la mise en place de la physiopathologie naturelle des NAFLD, notamment lors de la phase précoce de la maladie. Par ailleurs le projet a généré de la connaissance supplémentaire qui pourra être développée dans des projets ultérieurs afin d’établir des stratégies thérapeutiques ciblant le MI. Cet ensemble constitue un acquis important dans un domaine de recherche très compétitif qui génère un intérêt majeur de la part des industriels de l’agroalimentaires et des pharmaceutiques, et supporte ainsi le développement international des différents partenaires du projet.Non-alcoholic fatty liver diseases (NAFLD) include a spectrum of liver diseases ranging from fatty liver (NAFL), going through fibrosis (NASH), then cirrhosis in the absence of alcohol consumption, viral infection or other identified causes, and sometimes the development of hepatocellular carcinoma (HCC). Due to its high prevalence, up to a quarter of the general European population and its harmful consequences on the health of those who suffer from it, NAFLD represent a major public health issue.This liver disease has been associated with changes in gut microbiota and metabolic pathways in which this microbiota is involved. In mice, gut microbiota contributes to the worsening or protection of this disease.The goal of the thesis is to determine whether in humans the gut microbiota contributes also to this individual susceptibility observed in NAFLD mice.A complete standardized procedure for preparing, storing and managing human fecal transplants is developed and implemented (Burz et al., 2019).Specific pathogens free mice (SPF), pretreated with a cocktail of antibiotics, were colonized with microbiota derived from an healthy individual or patients at different stages of NAFLD. Then these mice were challenged with a high fructose, high fat diet in order to explore the effect of installed human microbiota on the onset and progression of induced NAFLD.This project made it possible to demonstrate, through the transfer of the human fecal microbiota, with constant food energy intake, the transfer of overweight. The human NAFL fecal microbiota worsens liver steatosis in mice, but mitigates liver and caecal inflammation.This thesis sheds additional light on the impact of gut microbiota in the establishment of the natural pathophysiology of NAFLD, especially during the early phase of the disease. The project also generated additional knowledge that could be developed in future projects in order to establish therapeutic strategies targeting gut microbiota. This set constitutes an important achievement in a very competitive research field, which generates major interest for food and pharmaceutical industries, and thus supports the international development of the different partners of the project

Alternative stable states in the intestinal ecosystem: proof of concept and a perspective of therapeutic implications

International audienc

Fecal microbiota transplant from human to mice gives insights into the role of the gut microbiota in non-alcoholic fatty liver disease (NAFLD)

International audienceNon-alcoholic fatty liver diseases (NAFLD) are associated with changes in the composition and metabolic activities of the gut microbiota. However, the causal role played by the gut microbiota in individual susceptibility to NAFLD and particularly at its early stage is still unclear. In this context, we transplanted the microbiota from a patient with fatty liver (NAFL) and from a healthy individual to two groups of mice. We first showed that the microbiota composition in recipient mice resembled the microbiota composition of their respective human donor. Following administration of a high-fructose, high-fat diet, mice that received the human NAFL microbiota (NAFLR) gained more weight and had a higher liver triglycerides level and higher plasma LDL cholesterol thanmice that received the human healthy microbiota (HR). Metabolomic analyses revealed that it was associated with lower and higher plasma levels of glycine and 3-Indolepropionic acid in NAFLR mice, respectively. Moreover, several bacterial genera and OTUs were identified as differently represented in the NAFLR and HR microbiota and therefore potentially responsible for the different phenotypes observed. Altogether, our results confirm that the gut bacteria play a role in obesity and steatosis development and that targeting the gut microbiota may be a preventive or therapeutic strategy in NAFLD management

The Efficacity of the NeuroAssist Robotic System for Motor Rehabilitation of the Upper Limb—Promising Results from a Pilot Study

The research aimed to evaluate the efficacy of the NeuroAssist, a parallel robotic system comprised of three robotic modules equipped with human–robot interaction capabilities, an internal sensor system for torque monitoring, and an external sensor system for real-time patient monitoring for the motor rehabilitation of the shoulder, elbow, and wrist. The study enrolled 10 consecutive patients with right upper limb paresis caused by stroke, traumatic spinal cord disease, or multiple sclerosis admitted to the Neurology I Department of Cluj-Napoca Emergency County Hospital. The patients were evaluated clinically and electrophysiologically before (T1) and after the intervention (T2). The intervention consisted of five consecutive daily sessions of 30–45 min each of 30 passive repetitive movements performed with the robot. There were significant differences (Wilcoxon signed-rank test) between baseline and end-point clinical parameters, specifically for the Barthel Index (53.00 ± 37.72 vs. 60.50 ± 36.39, p = 0.016) and Activities of Daily Living Index (4.70 ± 3.43 vs. 5.50 ± 3.80, p = 0.038). The goniometric parameters improved: shoulder flexion (70.00 ± 56.61 vs. 80.00 ± 63.59, p = 0.026); wrist flexion/extension (34.00 ± 28.75 vs. 42.50 ± 33.7, p = 0.042)/(30.00 ± 22.97 vs. 41.00 ± 30.62, p = 0.042); ulnar deviation (23.50 ± 19.44 vs. 33.50 ± 24.15, p = 0.027); and radial deviation (17.50 ± 18.14 vs. 27.00 ± 24.85, p = 0.027). There was a difference in muscle activation of the extensor digitorum communis muscle (1.00 ± 0.94 vs. 1.40 ± 1.17, p = 0.046). The optimized and dependable NeuroAssist Robotic System improved shoulder and wrist range of motion and functional scores, regardless of the cause of the motor deficit. However, further investigations are necessary to establish its definite role in motor recovery

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies