Search

Background & aimsFecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (CDI). However, transferring undefined living bacteria entails uncontrollable risks for infectious and metabolic or malignant diseases, particularly in immunocompromised patients. We investigated whether sterile fecal filtrates (containing bacterial debris, proteins, antimicrobial compounds, metabolic products, and oligonucleotides/DNA), rather than intact microorganisms, are effective in patients with CDI.MethodsWe performed a clinical case series to investigate the effects of fecal filtrate transfer (FFT) in 5 patients with symptomatic chronic-relapsing CDI at the Department of Internal Medicine I at the University Hospital Schleswig-Holstein (Kiel, Germany). Patients were followed up for at least 6 months and for up to 33 months. Stool was collected from 5 donors selected by the patients, and fully characterized according to FMT standards. Stool was sterile-filtered to remove small particles and bacteria; the filtrate was transferred to patients in a single administration via nasojejunal tube. Fecal samples were collected from patients before and at 1 week and 6 weeks after FFT. Microbiome, virome, and proteome profiles of donors and patients were compared.ResultsIn all 5 patients, FFT restored normal stool habits and eliminated symptoms of CDI for a minimum period of 6 months. Proteome analyses of selected FFT filtrates showed no obvious protein candidates associated with therapeutic efficacy. 16S ribosomal RNA gene sequencing detected diverse bacterial DNA signatures in the filtrates. Analysis of virus-like particles from a filtrate found to reduce symptoms of CDI showed a complex signature of bacteriophages. Bacterial phylogeny and virome profile analyses of fecal samples from recipients indicated longitudinal changes in microbial and viral community structures after FFT.ConclusionsA preliminary investigation of 5 patients with CDI shows that transfer of sterile filtrates from donor stool (FFT), rather than fecal microbiota, can be sufficient to restore normal stool habits and eliminate symptoms. This finding indicates that bacterial components, metabolites, or bacteriophages mediate many of the effects of FMT, and that FFT might be an alternative approach, particularly for immunocompromised patients.

Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell–cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes. [ABSTRACT FROM AUTHOR]

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease (1). Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction (2,3). As [Atg16l1.sup.HM] mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis (4). Unresolved ER stress is a common feature of inflammatory bowel disease epithelium (4,5), and several genetic risk factors of Crohn's disease affect Paneth cells (2,4,6-9). Here we show that impairment in either UPR ([Xbp1.sup.ΔIEC]) or autophagy function ([Atg16l1.sup.ΔIEC] or [Atg7.sup.ΔIEC]) in intestinal epithelial cells results in each other's compensatory engagement, and severe spontaneous Crohn's-disease-like transmural ileitis if both mechanisms are compromised. [Xbp1.sup.ΔIEC] mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumournecrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn's disease as a specific disorder of Paneth cells., The UPR and autophagy are integrally linked pathways (10). To investigate their relationship in the intestinal epithelium, we stably transduced the small intestinal epithelial cell line MODE-K with a short [...]

Analyzing the microbiome of diverse species and environments using next-generation sequencing techniques has significantly enhanced our understanding on metabolic, physiological and ecological roles of environmental microorganisms. However, the analysis of the microbiome is affected by experimental conditions (e.g. sequencing errors and genomic repeats) and computationally intensive and cumbersome downstream analysis (e.g. quality control, assembly, binning and statistical analyses). Moreover, the introduction of new sequencing technologies and protocols led to a flood of new methodologies, which also have an immediate effect on the results of the analyses. The aim of this work is to review the most important workflows for 16S rRNA sequencing and shotgun and long-read metagenomics, as well as to provide best-practice protocols on experimental design, sample processing, sequencing, assembly, binning, annotation and visualization. To simplify and standardize the computational analysis, we provide a set of best-practice workflows for 16S rRNA and metagenomic sequencing data (available at https://github.com/grimmlab/MicrobiomeBestPracticeReview). [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) printing is an innovative technology widely used in dentistry for manufacturing objects of various shapes using different materials. These advantages include enhanced predictability in oral rehabilitation, more effective treatment options, reduced chairside time, minimized human errors in the manufacturing process, shorter procedure durations, increased patient comfort, and the potential to enhance the treatment skills of practitioners. A dentist may recommend various types of removable or fixed space maintainers (SMs), including band and loop, crown and loop, distal end shoe, lingual arch, nance, and transpalatal arch, which are based on factors such as the number of lost teeth, stage of dental development, age of the child, and whether unilateral or bilateral tooth loss is present in the affected dental arch. The conventional manufacturing process of removable SMs (RSMs) is a technique-sensitive procedure, and ensuring the precision and fit of RSMs can be challenging due to the shrinkage of self-curing resin during polymerization. With the advent of CAD/CAM technology, manufacturing more accurate RSMs has become possible. These RSMs can be made from materials such as poly methyl methacrylate or acrylic resin, which are lightweight and provide stable results. In case of any growth-related changes, 3D-printed RSMs offer added flexibility as they can be easily trimmed, adjusted, relined, or even reprinted since digital files are readily available. This technology also enables digital documentation, eliminating the need for physical storage of patient data. [ABSTRACT FROM AUTHOR]