Jess L. Kaplan

Fecal Microbiota Transplant for Relapsing Clostridium difficile Infection Using a Frozen Inoculum From Unrelated Donors: A Randomized, Open-Label, Controlled Pilot Study

BACKGROUND Recurrent Clostridium difficile infection (CDI) with poor response to standard antimicrobial therapy is a growing medical concern. We aimed to investigate the outcomes of fecal microbiota transplant (FMT) for relapsing CDI using a frozen suspension from unrelated donors, comparing colonoscopic and nasogastric tube (NGT) administration. METHODS Healthy volunteer donors were screened and a frozen fecal suspension was generated. Patients with relapsing/refractory CDI were randomized to receive an infusion of donor stools by colonoscopy or NGT. The primary endpoint was clinical resolution of diarrhea without relapse after 8 weeks. The secondary endpoint was self-reported health score using standardized questionnaires. RESULTS A total of 20 patients were enrolled, 10 in each treatment arm. Patients had a median of 4 (range, 2-16) relapses prior to study enrollment, with 5 (range, 3-15) antibiotic treatment failures. Resolution of diarrhea was achieved in 14 patients (70%) after a single FMT (8 of 10 in the colonoscopy group and 6 of 10 in the NGT group). Five patients were retreated, with 4 obtaining cure, resulting in an overall cure rate of 90%. Daily number of bowel movements changed from a median of 7 (interquartile range [IQR], 5-10) the day prior to FMT to 2 (IQR, 1-2) after the infusion. Self-ranked health score improved significantly, from a median of 4 (IQR, 2-6) before transplant to 8 (IQR, 5-9) after transplant. No serious or unexpected adverse events occurred. CONCLUSIONS In our initial feasibility study, FMT using a frozen inoculum from unrelated donors is effective in treating relapsing CDI. NGT administration appears to be as effective as colonoscopic administration. CLINICAL TRIALS REGISTRATION NCT01704937.

The Role of Microbes in Developmental Immunologic Programming

The role of microorganisms in the gastrointestinal tract has undergone significant modification in the past few decades with new observations from clinical, epidemiologic, and basic science research. We now know that the perception of these gut microbes as pathogens or even as commensals is somewhat outdated. It is becoming increasingly clear that the gut microbiome plays an important role in a host of activities including digestion, protection from potentially pathogenic organisms, and the regulation and development of the host immune system. The complex interactions between microbes and host combined with recent clinical observations and epidemiologic trends may point to the convergence of two well-supported (though imperfect) hypotheses: the “hygiene hypothesis” and the “fetal programming hypothesis.” We are beginning to understand that exposure to microbes before conception, during gestation, and in the neonatal period have profound effects on the developing immune system. Recent observations from a variety of fields help support the expansion of the “fetal programming hypothesis” to a host-microbe corollary that microbe-host interactions at critical windows influence the future immune phenotype, the maintenance of immune health, and the development of immune-mediated disease.

Fecal Bacteriotherapy for Relapsing Clostridium difficile Infection in a Child: A Proposed Treatment Protocol

Clostridium difficile infection (CDI) is a potentially serious emerging infectious disease. The incidences of CDI in childhood and CDI cases complicated by relapses have increased by 50% or more in North America during the past 2 decades. We report here the case of a 2-year-old child with relapsing CDI caused by the epidemic strain BI/NAP1/O27 that was refractory to Saccharomyces boulardii and Lactobacillus rhamnosus GG probiotics and to intensive therapy with traditional (metronidazole, vancomycin) and experimental (rifaximin, nitazoxanide) antibiotics despite its apparent antimicrobial-susceptible phenotype. After excluding other infectious causes of diarrhea and inflammatory bowel disease, we designed a protocol to safely administer fecal bacteriotherapy via a temporary nasogastric tube. We demonstrated for the first time that fecal transplantation is practical and effective for treating relapsing CDI in a young child. We recommend that this strategy be reserved for complicated cases of CDI that fail conventional therapy until randomized studies can confirm the safety and effectiveness of fecal bacteriotherapy in children.

Fecal transplant for recurrent Clostridium difficile infection in children with and without inflammatory bowel disease.

ABSTRACT Ten children at our institution received single-infusion fecal microbiome transplant (FMT) using healthy, related screened donor stool to treat recurrent Clostridium difficile infection (RCDI) via nasogastric tube (2 patients) or colonoscopic delivery. Nine of the 10 (90%) children had resolution of their symptoms after a single-infusion FMT with follow-up of 1 month to 4 years. No concerning related adverse events were recognized during short- or long-term follow-up. Three of these children had concomitant inflammatory bowel disease and 2 of these 3 (66%) patients cleared RCDI with no clinical change in their underlying inflammatory bowel disease clinical activity as assessed by Physicians Global Assessment. All of the patients who had clinical improvement of gastrointestinal symptoms of RCDI while treated with antibiotics had lasting return of baseline health after FMT.

Duodenal helminth infection alters barrier function of the colonic epithelium via adaptive immune activation.

ABSTRACT Chronic infection with intestinal helminth parasites is a major public health problem, particularly in the developing world, and can have significant effects on host physiology and the immune response to other enteric infections and antigens. The mechanisms underlying these effects are not well understood. In the current study, we investigated the impact of infection with the murine nematode parasite Heligmosomoides polygyrus, which resides in the duodenum, on epithelial barrier function in the colon. We found that H. polygyrus infection produced a significant increase in colonic epithelial permeability, as evidenced by detection of elevated serum levels of the tracer horseradish peroxidase following rectal administration. This loss of normal barrier function was associated with clear ultrastructural changes in the tight junctions of colonic epithelial cells and an alteration in the expression and distribution of the junctional protein E-cadherin. These parasite-induced abnormalities were not observed in SCID mice but did occur in SCID mice that were adoptively transferred with wild-type T cells, indicating a requirement for adaptive immunity. Furthermore, the helminth-induced increase in gut permeability was not seen in STAT6 knockout (KO) mice. Taken together, the results demonstrate that one of the mechanisms by which helminths exert their effects involves the lymphocyte- and STAT6-dependent breakdown of the intestinal epithelial barrier. This increase in epithelial permeability may facilitate the movement of lumenal contents across the mucosa, thus helping to explain how helminth infection can alter the immune response to enteric antigens.

Helminth Infection Impairs Autophagy-Mediated Killing of Bacterial Enteropathogens by Macrophages

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.

A MicroRNA signature in pediatric ulcerative colitis: Deregulation of the miR-4284/CXCL5 pathway in the intestinal epithelium

Background:Twenty to 25% of the patients with inflammatory bowel disease (IBD) present the disease before the age of 18 to 20, with worse extent and severity, compared with adult-onset IBD. We sought to identify the differential expression of microRNAs in pediatric ulcerative colitis (UC) and their association with different clinical phenotypes. Methods:MicroRNA expression analysis was performed in colonic tissues derived from pediatric patients with UC and controls without IBD. MiR-4284 levels were verified by real-time quantitative polymerase chain reaction in 2 additional cohorts of pediatric patients with UC. Bioinformatics analysis was performed to predict the targets of miR-4284. In vitro experiments using luciferase reporter assays and real-time polymerase chain reaction evaluated the direct effect of miR-4284 on CXCL5 mRNA. In vivo experiments were performed in 2 mouse models of experimental colitis. Results:A 24-microRNA signature was identified in colonic tissues derived from pediatric patients with UC. The most downregulated microRNA in the tissue of pediatric patients UC, relative to non-IBD controls, was miR-4284. In situ hybridization revealed that miR-4284 is present in colonic epithelial cells, and its levels correlate with the disease activity. Furthermore, we found that miR-4284 regulates CXCL5 mRNA expression through binding to its 3′UTR. CXCL5 had increased mRNA levels in colonic tissue from pediatric patients with UC and correlated with disease activity. Furthermore, we found an inverse correlation between miR-4284 and CXCL5 levels in the colonic pediatric UC tissues and in 2 mouse models of experimental colitis. Conclusions:Our data reveal a novel microRNA pediatric UC signature and provide evidence that miR-4284 directly regulates CXCL5 and correlates with the disease activity.

DNA methylation-associated colonic mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.

Early gut colonization and subsequent obesity risk.

Purpose of reviewEarly microbial colonization patterns of the human gastrointestinal tract are increasingly implicated in the pathogenesis of human disease. Recently, large-scale shifts in gut microbiota have been demonstrated in both animal and human models of obesity. This review examines the latest research into the gut dysbiosis associated with an obese phenotype and considers the evidence that may link early microbial colonization patterns with subsequent obesity risk. Recent findingsStudies that link microbiome modifying early life events to subsequent obesity risk provide some indirect evidence to support a causal role for gut microbiota in the pathogenesis of obesity. However, more direct evidence proving causation is currently lacking and there is no existing support for the role of specific early gut colonization patterns in later risk of obesity. SummaryAlthough an obesity-associated dysbiosis is well supported by the current literature, cause and effect remain difficult to discern. Longitudinal, prospective studies that evaluate changes in gut microbial ecology over time are needed to better discern the role of specific microbial patterns in the pathogenesis of obesity. Better understanding of this relationship may lead to exciting new obesity treatment and prevention strategies in the future.

Microbiota separation and C-reactive protein elevation in treatment-naive pediatric granulomatous Crohn disease.

Objectives: In patients with inflammatory bowel diseases (IBDs), the presence of noncaseating mucosal granuloma is sufficient for diagnosing Crohn disease (CD) and may represent a specific immune response or microbial-host interaction. The cause of granulomas in CD is unknown and their association with the intestinal microbiota has not been addressed with high-throughput methodologies. Methods: The mucosal microbiota from 3 different pediatric centers was studied with 454 pyrosequencing of the bacterial 16S rRNA gene and the fungal small subunit (SSU) ribosomal region in transverse colonic biopsy specimens from 26 controls and 15 treatment-naïve pediatric CD cases. Mycobacterium avium subspecies paratuberculosis (MAP) was tested with real-time polymerase chain reaction. The correlation of granulomatous inflammation with C-reactive protein was expanded to 86 treatment-naïve CD cases. Results: The CD microbiota separated from controls by distance-based redundancy analysis (P = 0.035). Mucosal granulomata found in any portion of the intestinal tract associated with an augmented colonic bacterial microbiota divergence (P = 0.013). The granuloma-based microbiota separation persisted even when research center bias was eliminated (P = 0.04). Decreased Roseburia and Ruminococcus in granulomatous CD were important in this separation; however, principal coordinates analysis did not reveal partitioning of the groups. CRP levels >1 mg/dL predicted the presence of mucosal granulomata (odds ratio 28 [6–134.32]; 73% sensitivity, 91% specificity). Conclusions: Granulomatous CD associates with microbiota separation and C-reactive protein elevation in treatment-naïve children; however, overall dysbiosis in pediatric CD appears rather limited. Geographical/center bias should be accounted for in future multicenter microbiota studies.