Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Modulation of the microbiota by oral antibiotics treats immunoglobulin A nephropathy in humanized mice.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. IgA is mainly produced by the gut-associated lymphoid tissue (GALT). Both experimental and clinical data suggest a role of the gut microbiota in this disease. We aimed to determine if an intervention targeting the gut microbiota could impact the development of disease in a humanized mouse model of IgAN, the α1KI-CD89Tg mice. METHODS: Four- and 12-week old mice were divided into two groups to receive either antibiotics or vehicle control. Faecal bacterial load and proteinuria were quantified both at the beginning and at the end of the experiment, when blood, kidneys and intestinal tissue were collected. Serum mouse immunoglobulin G (mIgG) and human immunoglobulin A1 (hIgA1)-containing complexes were quantified. Renal and intestinal tissue were analysed by optical microscopy after haematoxylin and eosin colouration and immunohistochemistry with anti-hIgA and anti-mouse CD11b antibodies. RESULTS: Antibiotic treatment efficiently depleted the faecal microbiota, impaired GALT architecture and impacted mouse IgA production. However, while hIgA1 and mIgG serum levels were unchanged, the antibiotic treatment markedly prevented hIgA1 mesangial deposition, glomerular inflammation and the development of proteinuria. This was associated with a significant decrease in circulating hIgA1-mIgG complexes. Notably, final faecal bacterial load strongly correlated with critical clinical and pathophysiological features of IgAN such as proteinuria and hIgA1-mIgG complexes. In addition, treatment with broad-spectrum antibiotics reverted established disease. CONCLUSIONS: These data support an essential role of the gut microbiota in the generation of mucosa-derived nephrotoxic IgA1 and in IgAN development, opening new avenues for therapeutic approaches in this disease.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor.

BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Identification of NF-κB modulation capabilities within human intestinal commensal bacteria.

The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1ß, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations.

Parasites and diet as main drivers of the Malagasy gut microbiome richness and function.

Interactions between the prokaryotic microbiome and eukaryotic parasites in the vertebrate gut may affect overall host health and disease. While intertropical areas exhibit a high rate of parasites carriers, such interactions are understudied in these populations. Our objectives were to (1) describe the gut microbiome of individuals living in Madagascar, (2) identify potential associations between bacterial taxa and parasites colonizing the digestive tract and (3) highlight main determinants of the gut microbiota composition in this developing country. Metadata (socioeconomic, diet, clinical) and fecal samples were collected from 219 volunteers from North-West Madagascar (Mahajanga). Fecal microbiome was assessed through 16S rRNA gene sequencing and metabolomics, and related to dietary habits and parasites carriage. We highlight important Malagasy gut microbiome peculiarities. Out of three detected enterotypes, only one is similar to that observed in Westernized countries (Ruminococcus-driven). Functions associated with the two others (Clostridium sensu stricto-driven and Escherichia/Shigella-driven) are mostly directed toward amino acids biosynthesis and degradation, respectively. Diet and protozoan carriage were the main drivers of microbiota composition. High protozoan carriage was associated with higher diversity, richness and microbial functionalities. The gut microbiome of Malagasy strongly differs from that of Westernized countries. Asymptomatic protozoan carriage and dietary habits are the external factors with the deepest impact on gut microbiome. Further studies are needed to understand whether gut microbial richness constitute a predilection niche for protozoans colonization, due to their gazing features, or whether the parasites themselves induce a higher bacterial richness.

The clinical Pseudomonas fluorescens MFN1032 strain exerts a cytotoxic effect on epithelial intestinal cells and induces Interleukin-8 via the AP-1 signaling pathway.

BACKGROUND: Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response. RESULTS: Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-kappaB pathway. CONCLUSIONS: The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients.

Pickering emulsions with ethiodized oil and nanoparticles for slow release of intratumoral anti-CTLA4 immune checkpoint antibodies.

BACKGROUND: Intratumorous immunotherapy for cancer is currently thriving. The aim of such local strategy is to improve the therapeutic index of these treatments, for higher on-target/on-tumor activity and less on-target/off-tumor adverse events. Strategies allowing for slow release of anti-CTLA4 in the tumor microenvironment could improve their clinical efficacy.The purpose of the study was to develop a radiopaque delivery platform to improve the targeting and exposure of intratumorous anti-CTLA4 antibodies for cancer immunotherapy. METHODS: Pickering emulsions of anti-CTLA4 antibodies were formulated with radiopaque ethiodized oil and poly-lactic-co-glycolic acid (PLGA) nanoparticles. We characterized the microscopic aspect and stability of such emulsions using Turbiscan. We monitored the release of anti-CTLA4 over time from these emulsions and evaluated their structure using mass spectrometry. We then tested the functionality of the released antibodies by preforming ex vivo competitive binding assays. Finally, we assessed the in vivo efficacy of intratumorous anti-CTLA4 Pickering emulsions. RESULTS: Pickering emulsions of ethiodized oil and PLGA nanoparticles (PEEPs) resulted in a radiopaque water-in-oil emulsion with average internal phase droplet size of 42±5 µm at day 7. Confocal microscopy showed that anti-CTLA4 antibodies were effectively encapsulated by ethiodized oil with PLGA nanoparticles located at the interface between the aqueous and the oily phase. Turbiscan analysis showed that emulsions were stable with continuous and progressive release of anti-CTLA4 antibodies reaching 70% at 3 weeks. Structural and functional analysis of the released antibodies did not show significant differences with native anti-CTLA4 antibodies. Finally, intratumorous anti-CTLA4 PEEPs were able to eradicate tumors and cure mice in a syngeneic immunocompetent preclinical tumor model. CONCLUSION: Pickering emulsions of ethiodized oil and PLGA is an innovative radiopaque delivery platform that does not alter the functionality of anti-CTLA4 immune checkpoint antibodies. Beyond local anti-CTLA4 applications, these emulsions might be used with other therapeutic molecules for optimal intratumorous or intra-arterial delivery of novel cancer immunotherapies.

Assessment of Neonatal Intensive Care Unit Practices and Preterm Newborn Gut Microbiota and 2-Year Neurodevelopmental Outcomes.

Importance: In very preterm newborns, gut microbiota is highly variable with major dysbiosis. Its association with short-term health is widely studied, but the association with long-term outcomes remains unknown. Objective: To investigate in preterm newborns the associations among practice strategies in neonatal intensive care units (NICUs), gut microbiota, and outcomes at 2 years. Design, Setting, and Participants: EPIFLORE is a prospective observational cohort study that includes a stool sample collection during the fourth week after birth. Preterm newborns of less than 32 weeks of gestational age (GA) born in 2011 were included from 24 NICUs as part of the French nationwide population-based cohort, EPIPAGE 2. Data were collected from May 2011 to December 2011 and analyzed from September 2016 to December 2018. Exposures: Eight NICU strategies concerning sedation, ventilation, skin-to-skin practice, antibiotherapy, ductus arteriosus, and breastfeeding were assessed. A NICU was considered favorable to a practice if the percentage of that practice in the NICU was more than the expected percentage. Main Outcomes and Measures: Gut microbiota was analyzed by 16S ribosomal RNA gene sequencing and characterized by a clustering-based method. The 2-year outcome was defined by death or neurodevelopmental delay using a Global Ages and Stages questionnaire score. Results: Of 577 newborns included in the study, the mean (SD) GA was 28.3 (2.0) weeks, and 303 (52.5%) were male. Collected gut microbiota was grouped into 5 discrete clusters. A sixth cluster included nonamplifiable samples owing to low bacterial load. Cluster 4 (driven by Enterococcus [n = 63]), cluster 5 (driven by Staphylococcus [n = 52]), and cluster 6 (n = 93) were significantly associated with lower mean (SD) GA (26.7 [1.8] weeks and 26.8 [1.9] weeks, respectively) and cluster 3 (driven by Escherichia/Shigella [n = 61]) with higher mean (SD) GA (29.4 [1.6] weeks; P = .001). Cluster 3 was considered the reference. After adjustment for confounders, no assisted ventilation at day 1 was associated with a decreased risk of belonging to cluster 5 or cluster 6 (adjusted odds ratio [AOR], 0.21 [95% CI, 0.06-0.78] and 0.19 [95% CI, 0.06-0.62], respectively) when sedation (AOR, 10.55 [95% CI, 2.28-48.87] and 4.62 [1.32-16.18], respectively) and low volume of enteral nutrition (AOR, 10.48 [95% CI, 2.48-44.29] and 7.28 [95% CI, 2.03-26.18], respectively) was associated with an increased risk. Skin-to-skin practice was associated with a decreased risk of being in cluster 5 (AOR, 0.14 [95% CI, 0.04-0.48]). Moreover, clusters 4, 5, 6 were significantly associated with 2-year nonoptimal outcome (AOR, 6.17 [95% CI, 1.46-26.0]; AOR, 4.53 [95% CI, 1.02-20.1]; and AOR, 5.42 [95% CI, 1.36-21.6], respectively). Conclusions and Relevance: Gut microbiota of very preterm newborns at week 4 is associated with NICU practices and 2-year outcomes. Microbiota could be a noninvasive biomarker of immaturity.

Multimodal imaging for tumour characterization from micro- to macroscopic level using a newly developed dorsal chamber designed for long-term follow-up.

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One-shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow-up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long-term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long-term biological processes, as in cancer research.

Cord blood-endothelial colony forming cells are immunotolerated and participate at post-ischemic angiogenesis in an original dorsal chamber immunocompetent mouse model.

BACKGROUND: Cardiovascular diseases are the main cause of morbidity and mortality worldwide. Restoring blood supply to ischemic tissues is an essential goal for the successful treatment of these diseases. Growth factor or gene therapy efficacy remains controversial, but stem cell transplantation is emerging as an interesting approach to stimulate angiogenesis. Among the different stem cell populations, cord blood-endothelial progenitor cells (CB-EPCs) and more particularly cord blood-endothelial progenitor cell-derived endothelial colony forming cells (CB-ECFCs) have a great proliferative potential without exhibiting signs of senescence. Even if it was already described that CB-ECFCs were able to restore blood perfusion in hind-limb ischemia in an immunodeficient mouse model, until now, the immunogenic potential of allogenic CB-ECFCs remains controversial. Therefore, our objectives were to evaluate the immune tolerance potency of CB-ECFCs and their capacity to restore a functional vascular network under ischemic condition in immunocompetent mice. METHODS: In vitro, the expression and secretion of immunoregulatory markers (HLA-G, IL-10, and TGF-ß1) were evaluated on CB-ECFCs. Moreover, CB-ECFCs were co-cultured with activated peripheral blood mononuclear cells (PBMCs) for 6 days. PBMC proliferation was evaluated by [3H]-thymidine incorporation on the last 18 h. In vivo, CB-ECFCs were administered in the spleen and muscle of immunocompetent mice. Tissues were collected at day 14 after surgery. Finally, CB-ECFCs were injected intradermally in C57BL/6JRj mice close to ischemic macrovessel induced by thermal cauterization. Mice recovered until day 5 and were imaged, twice a week until day 30. RESULTS: Firstly, we demonstrated that CB-ECFCs expressed HLA-G, IL-10, and TGF-ß1 and secreted IL-10 and TGF-ß1 and that they could display immunosuppressive properties in vitro. Secondly, we showed that CB-ECFCs could be tolerated until 14 days in immunocompetent mice. Thirdly, we revealed in an original ischemic model of dorsal chamber that CB-ECFCs were integrated in a new functional vascular network. CONCLUSION: These results open up new perspectives about using CB-ECFCs as an allogeneic cell therapy product and gives new impulse to the treatment of cardiovascular diseases.

Towards a clinical application of freeze-dried squalene-based nanomedicines.

Squalene-adenosine (SQAd) nanoparticles (NPs) were found to display promising pharmacological activity similar to many other nanomedicines, but their long-term stability was still limited, and their preparation required specific know-how and material. These drawbacks represented important restrictions for their potential use in the clinic. Freeze-drying nanoparticles is commonly presented as a solution to allow colloidal stability, but this process needs to be adapted to each nanoformulation. Hence, we aimed at developing a specific protocol for freeze-drying SQAd NPs while preserving their structural features. NPs were lyophilised, resuspended and analysed by dynamic light scattering, atomic force microscopy and small-angle scattering. Among four different cryoprotectants, trehalose was found to be the most efficient in preserving NPs physico-chemical characteristics. Interestingly, we identified residual ethanol in NP suspensions as a key parameter which could severely affect the freeze-drying outcome, leading to NPs aggregation. Long-term stability was also assessed. No significant change in size distribution or zeta potential could be detected after three-month storage at 4 °C. Finally, freeze-dried NPs innocuity was checked in vitro on cultured hepatocytes and in vivo on mice. In conclusion, optimisation of freeze-drying conditions resulted in safe lyophilised SQAd NPs that can be easily stored, shipped and simply reconstituted into an injectable form.

A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation.

Owing 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.

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp.

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.

Identification of a group of shigella-like isolates as Shigella boydii 20.

Infections by Shigella species are an important cause of diarrhoeal disease worldwide. Of 4198 Shigella isolates received by the French National Reference Centre for Escherichia coli and Shigella, 180 from patients with diarrhoea and dysentery in 2000-2004 did not react with any available polyclonal rabbit antisera used to identify the established Shigella serogroups. This study describes the molecular and phenotypic characteristics of these isolates in seroagglutination tests, molecular serotyping (rfb-RFLP and fliC-RFLP), ribotyping, detection of invasivity and enterotoxins genes, and antibiotic sensitivity. All isolates gave biochemical reactions typical of Shigella boydii, were mannitol-positive and indole-negative. They all carried invasion-associated genes, enterotoxin 2 [ShET-2] and an IS630 sequence. They had a unique ribotype that was distinct from all other Shigella and E. coli patterns. Further characterization by rfb-RFLP clearly distinguished this serogroup from all other Shigella or E. coli O-groups. The fliC-RFLP pattern corresponded to P4, an F-pattern which is associated with 10 different serogroups of S. boydii. A new antiserum prepared against strain 00-977 agglutinated all 180 isolates and cross-agglutination and absorption studies with anti-00-977 serum and anti-CDC 99-4528 (reference for the newly described S. boydii serogroup 20) serum showed identical antigenic structure. Furthermore, strains 00-977 and CDC 99-4528 had the same molecular serotype, ribotype and virulence genes.

Respective Roles of Hematopoietic and Nonhematopoietic Nod2 on the Gut Microbiota and Mucosal Homeostasis.

BACKGROUND: NOD2 mutations are associated with Crohn's disease (CD). Both CD (in human) and Nod2 deficiency (in mice) are characterized by increased mucosal CD4 T-cells, an altered permeability and a microbial dysbiosis. However, the respective roles of the gut epithelial and immune compartments on the phenotype are not known. METHODS: Microbial composition, epithelial peptide secretion, intestinal permeability, and immune cell composition of Peyer patches were studied in Nod2 knock-out mice transplanted with wild-type bone marrow cells and vice versa. RESULTS: The nonhematopoietic cells control the microbiota composition and epithelial secretion of mucins and antimicrobial peptides. These parameters are correlated with recurrent associations between bacterial species and luminal products. In contrast, Nod2 in the hematopoietic compartment regulates the epithelial permeability and the gut-associated lymphoid tissue independently of the bacterial composition. CONCLUSIONS: The immune system and the gut permeability in one hand and the microbial and epithelial peptide compositions in the other hand are separate couples of interdependent parameters, both controlled by Nod2 in either the hematopoietic or nonhematopoietic lineages.

Nod2 Deficiency Leads to a Specific and Transmissible Mucosa-associated Microbial Dysbiosis Which Is Independent of the Mucosal Barrier Defect.

BACKGROUND AND AIMS: Crohn's disease [CD] is a complex disorder characterised by an inappropriate immune response, impaired barrier function and microbial dysbiosis. Mutations in nucleotide oligomeriation domain 2 [NOD2] are CD risk factors. Increase of intestinal permeability, CD4+ T cell infiltration, and bacterial dysbiosis are also seen in Nod2-knockout [Nod2 KO] mice. However, the specificity and relationship between these Nod2-associated abnormalities remain largely unexplored. METHODS: Wild-type [WT], Nod1-knockout [Nod1 KO] and Nod2 KO mice were analysed in parallel. Microbial composition was defined by 454-pyrosequencing of bacterial 16S rRNA genes. Mucin and antimicrobial peptide expression was assessed by RT-PCR. Cell populations from Peyer's patches were determined by flow cytometry. Ussing chambers were used to measure intestinal permeability and bacterial translocation. Finally, to explore the impact of colonisation with mother's microbiota at birth, analyses were also performed in Nod2 KO and WT mice born from WT surrogate mothers after embryo transfer. RESULTS: Nod2 KO mice exhibited colonic bacterial dysbiosis different from WT and Nod1 KO mice. Altered expression of antimicrobial peptides and mucins in ileum and colon was associated with the microbial composition. Bacterial composition of Nod2 KO and WT mice obtained by embryo transfer was similar to that observed in Nod2 KO mice, arguing for a dominant effect of Nod2 KO-associated dysbiosis. In contrast, increased levels of CD4+ T cells and gut barrier defects across Peyer's patches were specific to Nod2 deficiency and independent of Microbial dysbiosis. CONCLUSIONS: Nod2 deficiency is associated with a specific dominant dysbiosis which does not drive mucosal tissue and immune alterations.

Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells.

The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells.

Intrathecal synthesis of immunoglobulins in patients with unexplained intermediate uveitis.

PURPOSE: To assess the value of lumbar punctures in adult patients with unexplained intermediate uveitis (IU). METHODS: Retrospective study of 17 patients with unexplained IU. All the patients underwent physical examination, complete laboratory tests, and cerebrospinal MRI. RESULTS: Out of the 12 patients who underwent a lumbar puncture, six had oligoclonal bands and/or increased IgG index. CONCLUSION: Intrathecal synthesis of immunoglobulins is not rare in patients with unexplained IU. A longer follow-up is mandatory to determine whether intrathecal immunoglobulins synthesis has a predictive value for subsequent progression to multiple sclerosis.