Changes to the upper gastrointestinal microbiotas of children with reflux oesophagitis and oesophageal metaplasia.

Little is known of the relationships among paediatric upper gastrointestinal microbiotas, and the impact of medication use and disease on their diversity. Here, we investigated the diversity of three microbiotas in the upper gastrointestinal tract of paediatric patients in relation to each other and to host factors. Oral, oesophageal and gastric microbiotas from a prospective paediatric cohort (n=54) were profiled using the 16S rRNA gene and ITS2 amplicon sequencing. 16S rRNA gene amplicon sequencing of oesophageal biopsies from a retrospective paediatric cohort (n=96) and shotgun metagenomics data from oesophageal brushings (n=88) were employed for genomic signature validation. Bacterial diversity and composition showed substantial differences across oral, oesophageal and gastric fluid samples that were not replicated for fungi, and the presence of reflux led to increased homogeneity in the bacterial component of these three microbiotas. The oral and oesophageal microbiotas were associated with age, sex, history of oesophageal atresia and presence of oesophageal metaplasia, with the latter characterized by Prevotella enrichment. Proton pump inhibitor use was associated with increased oral bacterial richness in the gastric fluid, and this correlated with increased levels of gastric pro-inflammatory cytokines. Profiling of oesophageal biopsies from a retrospective paediatric cohort confirmed an increased Prevotella prevalence in samples with metaplasia. Analysis of metagenome-derived oesophageal Prevotella melaninogenica genomes identified strain-specific features that were significantly increased in prevalence in samples with metaplasia. Prevotella enrichment is a signature associated with paediatric oesophageal metaplasia, and proton pump inhibitor use substantially alters the paediatric gastric microenvironment.

Campylobacter concisus upregulates PD-L1 mRNA expression in IFN-γ sensitized intestinal epithelial cells and induces cell death in esophageal epithelial cells.

Introduction: Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) and Barrett's esophagus (BE). Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that is used by tumor cells for immune evasion and has increased expression in patients with IBD and BE. We examined whether C. concisus upregulates PD-L1 expression in intestinal and esophageal epithelial cells. Methods: Human intestinal epithelial HT-29 cells and esophageal epithelial FLO-1 cells with and without interferon (IFN)-γ sensitization were incubated with C. concisus strains. The level of PD-L1 mRNA was quantified using quantitative real-time PCR. Cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis of HT-29 and FLO-1 cells were measured using caspase 3/7 assay. Results: We found that intestinal epithelial cells with IFN-γ sensitization incubated with C. concisus significantly upregulated PD-L1 expression and significantly increased the production of interleukin (IL)-8. Whereas, PD-L1 expression was significantly inhibited in IFN-γ sensitized FLO-1 cells incubated with C. concisus strains. Furthermore, FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains both had significantly higher levels of cell death. Conclusion: C. concisushas the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects.

Multi-omics of the esophageal microenvironment identifies signatures associated with progression of Barrett's esophagus.

BACKGROUND: The enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host. METHODS: Here, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest. RESULTS: Profiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression. CONCLUSIONS: We provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia.

Escherichia coli K12 Upregulates Programmed Cell Death Ligand 1 (PD-L1) Expression in Gamma Interferon-Sensitized Intestinal Epithelial Cells via the NF-κB Pathway.

Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein which is used by tumor cells for immune evasion. PD-L1 is upregulated in inflamed intestinal tissues. The intestinal tract is colonized by millions of bacteria, most of which are commensal bacterial species. We hypothesized that under inflammatory conditions, some commensal bacterial species contribute to increased PD-L1 expression in intestinal epithelium and examined this hypothesis. Human intestinal epithelial HT-29 cells with and without interferon (IFN)-γ sensitization were incubated with six strains of four enteric bacterial species. The mRNA and protein levels of PD-L1 in HT-29 cells were examined using quantitative real-time PCR and flow cytometry, respectively. The levels of interleukin (IL)-1ß, IL-18, IL-6, IL-8, and tumor necrosis factor (TNF)-α secreted by HT-29 cells were measured using enzyme-linked immunosorbent assay. Apoptosis of HT-29 cells was measured using a caspase 3/7 assay. We found that Escherichia coli K12 significantly upregulated both PD-L1 mRNA and protein in IFN-γ-sensitized HT-29 cells. E. coli K12 induced the production of IL-8 in HT-29 cells, however, IL-8 did not affect HT-29 PD-L1 expression. Inhibition of the nuclear factor-kappa B pathway significantly reduced E. coli K12-induced PD-L1 expression in HT-29 cells. The other two E. coli strains and two enteric bacterial species did not significantly affect PD-L1 expression in HT-29 cells. Enterococcus faecalis significantly inhibited PD-L1 expression due to induction of cell death. Data from this study suggest that some gut bacterial species have the potential to affect immune function under inflammatory conditions via upregulating epithelial PD-L1 expression.

Detection of IL-18 and IL-1ß protein and mRNA in human oral epithelial cells induced by Campylobacter concisus strains.

Campylobacter concisus is an emerging bacterial pathogen that may play a role in the development of inflammatory bowel disease and oral inflammatory conditions such as periodontal disease. To elucidate the role and pathogenic mechanisms of C. concisus in contributing to oral inflammation, this study examined the production of IL-1 family proinflammatory cytokines IL-18 and IL-1ß in oral epithelial cells induced by C. concisus strains using enzyme-linked immunosorbent assay (ELISA), Western-blot and quantitative real-time PCR. C. concisus increased the mRNA levels of IL-18 and IL-1ß in oral epithelial cells. Furthermore, a large amount of IL-18 in the supernatants of oral epithelial cells infected with C. concisus strains was detected by ELISA, and various experiments demonstrated that this positive signal was derived from C. concisus bacterium. The findings that C. concisus upregulated IL-18 and IL-1ß in oral epithelial cells from this study support a role of C. concisus in oral inflammatory diseases. Furthermore, the finding that C. concisus released a molecule that was strongly cross-reactive to anti-human IL-18 monoclonal antibodies suggests that in future studies examining cytokines induced by bacterial microbes, a bacterium control should be included.

Global Investigations of Fusobacterium nucleatum in Human Colorectal Cancer.

Colorectal cancer (CRC) is the third most prevalent cancer and second in terms of mortality. Emerging evidence from recent studies suggests a potential role of Fusobacterium nucleatum in the development of CRC. In this article, we review studies from different geographical regions examining the association between F. nucleatum and CRC, the detection methods and the tumorigenic mechanisms. Furthermore, we discuss the potential clinical impact of F. nucleatum in CRC and suggest future study directions.

Signatures within the esophageal microbiome are associated with host genetics, age, and disease.

BACKGROUND: The esophageal microbiome has been proposed to be involved in a range of diseases including the esophageal adenocarcinoma cascade; however, little is currently known about its function and relationship to the host. Here, the esophageal microbiomes of 106 prospectively recruited patients were assessed using 16S rRNA and 18S rRNA amplicon sequencing as well as shotgun sequencing, and associations with age, gender, proton pump inhibitor use, host genetics, and disease were tested. RESULTS: The esophageal microbiome was found to cluster into functionally distinct community types (esotypes) defined by the relative abundances of Streptococcus and Prevotella. While age was found to be a significant factor driving microbiome composition, bacterial signatures and functions such as enrichment with Gram-negative oral-associated bacteria and microbial lactic acid production were associated with the early stages of the esophageal adenocarcinoma cascade. Non-bacterial microbes such as archaea, Candida spp., and bacteriophages were also identified in low abundance in the esophageal microbiome. Specific host SNPs in NOTCH2, STEAP2-AS1, and NREP were associated with the composition of the esophageal microbiome in our cohort. CONCLUSIONS: This study provides the most comprehensive assessment of the esophageal microbiome to date and identifies novel signatures and host markers that can be investigated further in the context of esophageal adenocarcinoma development.

OPCML is hypermethylated in a subset of patients with metaplastic changes in their esophagus.

OPCML hypermethylation is considered a promising cancer biomarker. We examined methylation levels in the first exon of OPCML in two patient cohorts within the esophageal adenocarcinoma and gastric adenocarcinoma cascades and in a range of cell-lines using a custom PyroMark CpG assay. Methylation levels were significantly higher in esophageal tissue with histologically confirmed glandular mucosa as compared to tissue from normal esophagi or gastro-esophageal reflux disease. Higher levels of OPCML methylation were absent in the adjacent normal esophageal tissue of patients with glandular mucosa. Higher levels of methylation were confirmed in cell-lines derived from patients with adenocarcinoma, but also detected in two cell-lines with signs of dysplasia. We validated our assay by showing no differences in methylation levels in DNA extracted from blood of patients within the gastric adenocarcinoma cascade. OPCML hypermethylation is present in a subset of patients with metaplastic changes in their esophagus.

Genomic analysis of oral Campylobacter concisus strains identified a potential bacterial molecular marker associated with active Crohn's disease.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC). C. concisus consists of two genomospecies (GS) and diverse strains. This study aimed to identify molecular markers to differentiate commensal and IBD-associated C. concisus strains. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this study. We identified a novel secreted enterotoxin B homologue, Csep1. The csep1 gene was found in 56% of GS2 C. concisus strains, presented in the plasmid pICON or the chromosome. A six-nucleotide insertion at the position 654-659 bp in csep1 (csep1-6bpi) was found. The presence of csep1-6bpi in oral C. concisus strains isolated from patients with active CD (47%, 7/15) was significantly higher than that in strains from healthy controls (0/29, P = 0.0002), and the prevalence of csep1-6bpi positive C. concisus strains was significantly higher in patients with active CD (67%, 4/6) as compared to healthy controls (0/23, P = 0.0006). Proteomics analysis detected the Csep1 protein. A csep1 gene hot spot in the chromosome of different C. concisus strains was found. The pICON plasmid was only found in GS2 strains isolated from the two relapsed CD patients with small bowel complications. This study reports a C. concisus molecular marker (csep1-6bpi) that is associated with active CD.

Campylobacter concisus and inflammatory bowel disease.

Investigation of the possible role of Campylobacter concisus (C. concisus) in inflammatory bowel disease (IBD) is an emerging research area. Despite the association found between C. concisus and IBD, it has been difficult to explain how C. concisus, a bacterium that is commonly present in the human oral cavity, may contribute to the development of enteric diseases. The evidence presented in this review shows that some C. concisus strains in the oral cavity acquired zonula occludens toxin (zot) gene from a virus (prophage) and that C. concisus Zot shares conserved motifs with both Vibrio cholerae Zot receptor binding domain and human zonulin receptor binding domain. Both Vibrio cholerae Zot and human zonulin are known to increase intestinal permeability by affecting the tight junctions. Increased intestinal permeability is a feature of IBD. Based on these data, we propose that a primary barrier function defect caused by C. concisus Zot is a mechanism by which zot-positive C. concisus strains may trigger the onset and relapse of IBD.

Prevalence of Campylobacter species in adult Crohn's disease and the preferential colonization sites of Campylobacter species in the human intestine.

INTRODUCTION: Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). A high prevalence of Campylobacter concisus was previously detected in paediatric CD and adult UC. Currently, the prevalence of C. concisus in adult CD and the preferential colonization sites of Campylobacter species in the human intestine are unknown. In this study, we examined the prevalence of Campylobacter species in biopsies collected from multiple anatomic sites of adult patients with IBD and controls. METHODS: Three hundred and one biopsies collected from ileum, caecum, descending colon and rectum of 28 patients IBD (15 CD and 13 UC) and 33 controls were studied. Biopsies were used for DNA extraction and detection of Campylobacter species by PCR-sequencing and Campylobacter cultivation. RESULTS: A significantly higher prevalence of C. concisus in colonic biopsies of patients with CD (53%) was detected as compared with the controls (18%). Campylobacter genus-PCR positivity and C. concisus positivity in patients with UC were 85% and 77% respectively, being significantly higher than that in the controls (48% and 36%). C. concisus was more often detected in descending colonic and rectal biopsies from patients with IBD in comparison to the controls. C. concisus was isolated from patients with IBD. CONCLUSION: The high intestinal prevalence of C. concisus in patients with IBD, particularly in the proximal large intestine, suggests that future studies are needed to investigate the possible involvement of C. concisus in a subgroup of human IBD. To our knowledge, this is the first report of the association between adult CD and C. concisus as well as the first study of the preferential colonization sites of C. concisus in the human intestine.

Cystic fibrosis-related diabetes in childhood.

Since the early 1990s the management of children with cystic fibrosis (CF) has come a long way and advances in both nutritional and medical care have resulted in a median age of survival of 30-35 years, as compared with a life expectancy of <1 year in the 1950s. The first definitive reports of glucose intolerance or diabetes in CF are from 1955. The combination of CF and related diabetes (CFRD) has a negative impact on survival. CFRD is now the most common complication of CF (50% of the CF patients will develop diabetes by the age of 30 years), and is associated with a 6-fold increase in morbidity and mortality. CFRD is usually asymptomatic and can remain undetected for up to 4 years prior to diagnosis. The objective of this report was to review the current literature (Medline and Pubmed searches) on CFRD in children and adolescents and provide a comprehensive report of incidence, prevalence and pathophysiology of insulin deficiency and insulin insensitivity. Along with survival and prognosis in CFRD the current management strategies in the diagnosis, monitoring and treatment of CFRD will also be addressed.

Validation of continuous glucose monitoring in children and adolescents with cystic fibrosis: a prospective cohort study.

OBJECTIVE: To validate continuous glucose monitoring (CGM) in children and adolescents with cystic fibrosis. RESEARCH DESIGN AND METHODS: Paired oral glucose tolerance tests (OGTTs) and CGM monitoring was undertaken in 102 children and adolescents with cystic fibrosis (age 9.5-19.0 years) at baseline (CGM1) and after 12 months (CGM2). CGM validity was assessed by reliability, reproducibility, and repeatability. RESULTS: CGM was reliable with a Bland-Altman agreement between CGM and OGTT of 0.81 mmol/l (95% CI for bias +/- 2.90 mmol/l) and good correlation between the two (r = 0.74-0.9; P < 0.01). CGM was reproducible with no significant differences in the coefficient of variation of the CGM assessment between visits and repeatable with a mean difference between CGM1 and CGM2 of 0.09 mmol/l (95% CI for difference +/- 0.46 mmol/l) and a discriminant ratio of 13.0 and 15.1, respectively. CONCLUSIONS: In this cohort of children and adolescents with cystic fibrosis, CGM performed on two occasions over a 12-month period was reliable, reproducible, and repeatable.

Perspectives on liver failure: past and future.

Acute liver failure (ALF) remains a potentially devastating syndrome with a high mortality rate. Much interest continues to center upon the possibility of providing effective liver support through various artificial and bioartificial extracorporeal devices. The development of purpose-designed intensive care units for the treatment of ALF allowed a better understanding of the clinical syndrome and its best management, through the ability to conduct detailed clinical observation and clinical trials of new interventions. Survival rates are substantially improved today compared with the mortality rate that approximated 100% when the syndrome was first described nearly five decades ago. Nonetheless, these have plateaued in recent years, prompting one to consider whether major new advances in disease understanding are needed to further improve the overall outcome. A major challenge to a broader understanding of disease pathogenesis and the ability to direct appropriate therapy remains the substantial number of cases of ALF for which no specific etiology can be identified. Much is now known about the basic molecular mechanisms underlying hepatocyte cell death in ALF and an important issue is whether these cell death pathways can, in the future, be interrupted for therapeutic gain. Such considerations would seem most relevant in the early stages of the clinical course, when damage to the liver is not so severe but perhaps programmed to so evolve. A particular challenge will be to reduce hepatocellular death without inhibiting the liver's regenerative potential, given the pleiotropic functions of some of the molecules involved in both processes.

Lactose malabsorption in the elderly: role of small intestinal bacterial overgrowth.

OBJECTIVE: The prevalence of lactose malabsorption (LM) is increased in the elderly, although the mechanisms responsible are still a matter of speculation. The objective of this study was to investigate the possible roles of reduced functional small intestinal absorptive area, lactase deficiency and small intestinal bacterial overgrowth (SIBO). MATERIAL AND METHODS: Twenty Caucasian (Anglo-Celtic), asymptomatic, well-nourished, elderly volunteers (median age 79 years, range 70-94 years) with no clinically apparent predisposition to SIBO underwent a 50 g lactose breath hydrogen test (LBHT) and mannitol absorption test, the latter as an index of functional small intestinal absorptive area. Those with LM additionally underwent bacteriological assessment of small intestinal secretions and mucosal biopsy, to assess the contribution of SIBO and lactase deficiency, respectively, to the pathogenesis of LM in individual cases. The prevalence of SIBO was also determined in elderly subjects without LM. Twenty asymptomatic younger subjects (median age 29 years, age range 18-35 years) served as controls. All subjects were "hydrogen producers" in response to lactulose. RESULTS: LM was evident in 10/20 (50%) elderly subjects and 1/20 (5%) younger subjects (p=0.003). Mannitol absorption did not differ significantly in elderly and younger subjects or in elderly subjects with and without LM. SIBO was documented in 9/10 (90%) elderly subjects with LM; eradication was associated with resolution of LM. Lactase deficiency was evident in only one elderly subject with LM. SIBO was evident in 2/10 (20%) elderly subjects without LM (p=0.005 compared to those with LM). Lactulose breath hydrogen test identified only 2/11 (18%) elderly subjects with SIBO. CONCLUSIONS: Increased prevalence of LM in the elderly is mostly due to clinically non-apparent SIBO, rather than mucosal factors. The lactulose breath hydrogen test cannot be relied upon to identify elderly subjects with SIBO, even in those without an anatomical predisposition.

Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.

UNLABELLED: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production. CONCLUSION: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.