Coeliac disease is defined by gluten responsiveness, yet there are few data on gluten challenge (GC) in adults on a gluten free diet. Lack of data regarding the kinetics of responses to gluten is a limitation in clinical practice and research when GC is performed.
20 adults with biopsy-proven coeliac disease participated. The study included two run-in visits followed by a 14 day GC at a randomly assigned dose of 3 or 7.5 grams of gluten/day. Study visits occurred 3, 7, 14 and 28 days after starting GC. Duodenal biopsy was performed during the run-in and at days 3 and 14 of GC. Villous height to crypt depth ratio (Vh:Cd) and intraepithelial lymphocyte (IEL) count/100 enterocytes were measured by two pathologists. Antibodies to tTG and DGP, lactulose to mannitol ratio (LAMA), and symptoms were assessed at each visit.
Significant reduction in Vh:Cd (2.2 to 1.1, p < 0.001) and increase in IELs (32.6 to 51.8, p < 0.001) were seen from baseline to day 14. Antibody titers increased slightly from baseline to day 14 of GC but markedly by day 28. LAMA did not change significantly. Gastrointestinal symptoms increased significantly by day 3 and returned to baseline by day 28. No differences were seen between the two gluten doses.
14 day GC at ≥3 grams of gluten/day induces histological and serological changes in the majority of adults with coeliac disease. These data permit accurate design of clinical trials and indicate that many individuals will meet coeliac diagnostic criteria after a two week GC.
coeliac disease; gluten challenge; histology; transglutaminase; symptoms
To study the mechanism(s) linking insulin resistance (IR) to hepatic fibrosis and the role of the epithelial component in tissue repair and fibrosis in chronic hepatitis C (CHC).
Prospective observational study.
Tertiary care academic centre.
78 consecutive patients with CHC.
Main outcome measures
IR, calculated by the oral glucose insulin sensitivity during oral glucose tolerance test; necroinflammatory activity and fibrosis, defined according to Ishak’s score; steatosis, graded as 0 (<5% of hepatocytes), 1 (5–33%), 2 (33–66%) and 3 (>66%). To evaluate the role of the epithelial component in tissue repair and fibrosis, the expansion of the ductular reaction (DR) was calculated by keratin-7 (CK7) morphometry. Nuclear expression of Snail, downregulation of E-cadherin and expression of fibroblast specific protein-1 (FSP1) and vimentin by CK7-positive cells were used as markers of epithelial-mesenchymal transition in DR elements.
IR, the degree of necroinflammation and expansion of the DR (stratified as reactive ductular cells (RDCs), hepatic progenitor cells and intermediate hepatobiliary cells according to morphological criteria) were all associated with the stage of fibrosis. Nuclear Snail expression, E-cadherin downregulation and vimentin upregulation were observed in RDCs. By dual immunofluorescence for CK7 and FSP1, the number of RDCs undergoing epithelial-mesenchymal transition progressively increased together with the necroinflammatory score. By multivariate analysis, total inflammation and insulin resistance were the only factors significantly predicting the presence of advanced fibrosis (Ishak score ≥3) and the expansion of RDCs.
This study indicates that IR is associated with the degree of necroinflammatory injury in CHC and contributes to hepatic fibrosis by stimulating the expansion of RDCs that express epithelial-mesenchymal transition markers.
Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that Spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for Lgr5 in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia.
Lgr5-EGFP-IRES-CreERT2/+;Rosa26R mice were utilized to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L635 to induce acute SPEM. Lineage mapping was performed to determine if LGR5-expressing cells gave rise to SPEM.
Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-CreERT2/+;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed.
The results indicate that, while chief cells with Lgr5-transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.
SPEM; chief cell; Lgr5; TFF2; oxyntic atrophy
Genotype-specific associations between hepatitis C virus (HCV) and insulin resistance (IR) have been described, but a causal relationship remains unclear. This study investigated the association between a sustained virological response (SVR) and IR after chronic HCV therapy.
2255 treatment-naive patients with chronic HCV genotype 1 or 2/3 were enrolled in two phase 3 trials of albinterferon alpha-2b versus pegylated interferon alpha-2a for 48 or 24 weeks, respectively. IR was measured before treatment and 12 weeks after treatment using homeostasis model assessment (HOMA)-IR.
Paired HOMA-IR measurements were available in 1038 non-diabetic patients (497 with genotype 1; 541 with genotype 2/3). At baseline the prevalence of HOMA-IR >3 was greater in patients with genotype 1 than 2/3 (33% vs 27%; p=0.048). There was a significant reduction in the prevalence of IR in patients with genotype 1 achieving SVR (δ 10%; p<0.001), but not in genotype 1 non-responders or those with genotype 2/3. Multivariate analysis indicated that SVR was associated with a significant reduction in mean HOMA-IR in patients with genotype 1 (p=0.004), but not in those with genotype 2/3, which was independent of body mass index, alanine transaminase, γ-glutamyl transpeptidase and lipid level changes.
SVR is associated with a reduction in HOMA-IR in patients with HCV genotype 1 but not in those with genotype 2/3. Genotype 1 may have a direct effect on the development of IR, independent of host metabolic factors, and may be partially reversed by viral eradication.
Exposure of the oesophageal mucosa to gastric acid and bile acids leads to accumulation of reactive oxygen species (ROS), a known risk factor for Barrett’s oesophagus (BO) and progression to oesophageal adenocarcinoma (OAC). In this study we investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage.
Using in vitro cell models, we performed experiments that included GPX activity, Amplex UltraRed, CM-H2DCFDA, Annexin-V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and Western blot assays.
Enzymatic assays demonstrated a limited GPX activity of the recombinant GPX7 protein. However, GPX7 exhibited a strong capacity to neutralize H2O2 independent of glutathione. Reconstitution of GPX7 expression in immortalized BO cells, BAR-T and CP-A, led to resistance to H2O2-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH 4), these GPX7-expressing cells demonstrated lower levels of H2O2, intracellular ROS, oxidative DNA damage and double strand breaks (DSB), as compared to control (P<0.01). In addition, these cells demonstrated lower levels of ROS signaling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H2O2-induced oxidative stress. The knockdown of endogenous GPX7 in immortalized oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H2O2, intracellular ROS, oxidative DNA damage, DSB, apoptosis and ROS-dependent signaling (P<0.01).
The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage which are common risk factors for BO and OAC.
Glutathione peroxidase 7; Barrett’s; Oesophagus; Cancer; DNA damage; Bile acids; ROS
Immune responses are important in dictating nonalcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbor large numbers of NKT cells. Here, we evaluated the hypothesis that activated NKT cells drive fibrogenesis during NASH by assessing if NKT depletion protects against NASH-fibrosis; identifying the NKT associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. When fed methionine choline deficient (MCD) diets for 8 weeks, WT mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Jα18−/− and CD1d−/− mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNC) from MCD diet-fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated hepatic stellate cells (HSC) to become myofibroblasts (MF); neutralizing these factors abrogated the fibrogenic actions of WT LMNC. LMNC from NKT cell deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSC. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression, and correlated with fibrosis severity. In conclusion, hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH-fibrosis.
biomarker; fibrosis; natural killer T cells; non-alcoholic steatohepatitis; Spp1
Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties.
To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes.
Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP.
Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice.
Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.
Background and Aim
Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. Curcumin, the yellow pigment of the spice turmeric, has pleiotropic actions and attenuates hepatic damage in animal models of chemically-induced liver injury. Whether curcumin has beneficial effects in cholangiopathies is unknown.
Potential anti-cholestatic, anti-inflammatory and anti-fibrotic mechanisms of curcumin were explored in vivo in Mdr2−/− mice as a murine model of chronic cholangiopathy; as well as in vitro in a cholangiocyte cell line (HuCCT1) and portal myofibroblasts (MFBs) isolated from Mdr2−/− mice.
Liver damage, cholestasis and fibrosis were reduced in Mdr2−/− mice after curcumin feeding. Moreover, curcumin inhibited cholangiocyte proliferation and expression of activation marker Vascular Cell Adhesion Molecule-1 (VCAM-1) in Mdr2−/− mice. Curcumin - similar to PPARγ synthetic agonist troglitazone - directly inhibited TNF-α induced inflammatory activation of cholangiocytes in vitro, whereas these beneficial effects of curcumin were largely blocked by a PPARγ synthetic antagonist. In addition, curcumin blocked proliferation and activation of portal MFBs by inhibiting ERK1/2 phosphorylation, thus contributing to reduced fibrogenesis.
These results show that curcumin may have multiple targets in liver including activation of PPARγ in cholangiocytes and inhibition of ERK1/2 signalling in MFBs, thereby modulating several central cellular events in a mouse model of cholangiopathy. Targeting these pathways may be a promising therapeutic approach to cholangiopathies.
cholangiopathy; curcumin; Mdr2; portal fibrosis; sclerosing cholangitis
The aim of this study was to explore the association of serum fibrosis marker levels with the risk of clinical and histological disease progression in a large cohort of patients with chronic hepatitis C (CHC)
462 prior non-responders to peginterferon and ribavirin enrolled in the randomized phase of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial had baseline and annual serum samples tested for hyaluronic acid (HA), n-terminal peptide of procollagen type 3, tissue inhibitor of matrix metalloproteinase-1, and YKL-40.
All patients underwent a pretreatment liver biopsy and follow-up biopsies at years 2 and 4. Histological progression was defined as a ≥ 2 point increase in Ishak fibrosis score in non-cirrhotic patients. Clinical outcomes included development of decompensation, hepatocellular cancer, death, or an increase in the CTP score to ≥ 7.
Mean patient age was 49.5 years and 39% had histological cirrhosis at entry. Baseline HA, YKL-40 and TIMP-1 levels combined with other laboratory parameters were all significantly associated with clinical outcomes in the 69 (15%) patients with disease progression (p< 0.0001). The best multivariate model to predict clinical outcomes included baseline bilirubin, albumin, INR, and YKL-40 levels. All of the baseline serum fibrosis marker levels were also significantly associated with histological fibrosis progression that developed in 70 (33%) of the 209 non-cirrhotic patients (p < 0.0001). However, baseline HA and platelet counts were best at predicting histological progression (AUC = 0.663).
Pretreatment serum fibrosis marker levels are significantly increased in CHC patients at risk of clinical and histological disease progression. If validated in additional cohorts, measurement of these markers could help identify CHC patients who would benefit from more frequent and intensive monitoring.
Cirrhosis; non-invasive markers; liver fibrosis; natural history; viral hepatitis
Background and aims
Celiac disease (cd) is a common small intestinal inflammatory disorder that results from a breach of intestinal tolerance to dietary gluten proteins, driven by gluten-reactive effector T cells. We aimed to assess the pathogenic role of gluten-reactive effector T cells and to generate a model of gluten-induced enteropathy.
CD4+CD25− T cell fractions were adoptively transferred into lymphopenic mice, leading to “baseline” small intestinal inflammation.
Rag1−/− recipients of gliadin-presensitized CD4+CD45RBlowCD25− T cells, but not CD4+CD45RBhigh naive T cells, gained less weight and suffered from more severe duodenitis when challenged with oral gluten than recipients on gluten-free diet, or recipients of control (ovalbumin)-presensitized T cells. This was accompanied by deterioration of mucosal histological features characteristic of cd, and increased Th1/Th17 cell polarization in the duodenum and the periphery. Interestingly, reintroduction of a gluten-free diet led to weight gain, improvement of histological duodenitis, and a decrease in duodenal IFNγ and IL-17 transcripts. Moreover, B cell-competent nude recipients of gliadin-presensitized T cells produced high levels of serum anti-gliadin IgA and IgG1/IgG2c only when challenged with oral gluten.
CD4+ T cell immunity to gluten leads to a breach of oral gluten tolerance and small intestinal pathology in lymphopenic mice, similar to human cd. This model will be useful for the study of cd pathogenesis, but also for testing novel non-dietary therapies for cd.
Celiac disease; gluten-sensitive enteropathy; small intestine; duodenitis; enteritis; gliadin; gluten; memory T cells; regulatory T cells; CD4+CD45RBlowCD25− T cells; intestinal antigen; food protein; oral tolerance; mucosal immunology
Oral contraceptive use has been associated with risk of Crohn’s disease (CD) and ulcerative colitis (UC).
To determine whether this association is confounded or modified by other important lifestyle and reproductive factors.
A prospective cohort study was carried out of 117 375 US women enrolled since 1976 in the Nurses Health Study I (NHS I) and 115 077 women enrolled since 1989 in the Nurses’ Health Study II (NHS II) with no prior history of UC or CD. These women had provided information every 2 years, on age at menarche, oral contraceptive use, parity, menopause status and other risk factors. Diagnoses of CD and UC were confirmed by review of medical records. Cox proportional hazards models were used to calculate HRs and 95% CIs.
Among 232 452 women with over 5 030 196 person-years of follow-up, 315 cases of CD and 392 cases of UC were recorded through 2007 in NHS II and 2008 in NHS I. Compared with never users of oral contraceptives, the multivariate-adjusted HRs for CD were 2.82 (95% CI 1.65 to 4.82) among current users and 1.39 (95% CI 1.05 to 1.85) among past users. The association between oral contraceptives and UC differed according to smoking history (pheterogeneity = 0.04). Age at menarche, age at first birth and parity were not associated with risk of UC or CD.
In two large prospective cohorts of US women, oral contraceptive use was associated with risk of CD. The association between oral contraceptive use and UC was limited to women with a history of smoking.
The earliest endoscopically-evident lesion in Crohn’s disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn’s disease is poorly understood.
Using a mouse model of Crohn’s-like ileitis (TNFΔARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed.
Both CCL19 and CCL21 were increased within the inflamed ileum of TNFΔARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis.
Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19–CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.
The associations between oral diseases and increased risk of pancreatic cancer have been reported in several prospective cohort studies. In this study, we measured variations of salivary microbiota and evaluated their potential associations with pancreatic cancer and chronic pancreatitis.
This study was divided into three phases: (1) microbial profiling using the Human Oral Microbe Identification Microarray to investigate salivary microbiota variation between 10 resectable patients with pancreatic cancer and 10 matched healthy controls, (2) identification and verification of bacterial candidates by real-time quantitative PCR (qPCR) and (3) validation of bacterial candidates by qPCR on an independent cohort of 28 resectable pancreatic cancer, 28 matched healthy control and 27 chronic pancreatitis samples.
Comprehensive comparison of the salivary microbiota between patients with pancreatic cancer and healthy control subjects revealed a significant variation of salivary microflora. Thirty-one bacterial species/clusters were increased in the saliva of patients with pancreatic cancer (n=10) in comparison to those of the healthy controls (n=10), whereas 25 bacterial species/clusters were decreased. Two out of six bacterial candidates (Neisseria elongata and Streptococcus mitis) were validated using the independent samples, showing significant variation (p<0.05, qPCR) between patients with pancreatic cancer and controls (n=56). Additionally, two bacteria (Granulicatella adiacens and S mitis) showed significant variation (p<0.05, qPCR) between chronic pancreatitis samples and controls (n=55). The combination of two bacterial biomarkers (N elongata and S mitis) yielded a receiver operating characteristic plot area under the curve value of 0.90 (95% CI 0.78 to 0.96, p<0.0001) with a 96.4% sensitivity and 82.1% specificity in distinguishing patients with pancreatic cancer from healthy subjects.
The authors observed associations between variations of patients’ salivary microbiota with pancreatic cancer and chronic pancreatitis. This report also provides proof of salivary microbiota as an informative source for discovering non-invasive biomarkers of systemic diseases.
Trefoil factor (TFF) peptides are expressed in gastric tissues, where they are part of the epithelial defences. To complement previous in vitro work, the goal of the present study was to examine directly if TFF2 was essential for gastric restitution in vivo during the recovery from microscopic damage.
TFF2 mutant (KO) mice were examined to study the epithelial repair process in vivo after laser-induced photodamage (LPD). Using two-photon laser energy absorption (710 nm), LPD was imposed on an ~3–5 cell region of surface epithelium in anaesthetised mouse stomach. Responses to damage were evaluated during confocal time-lapse microscopy; including area of damage and the extracellular pH adjacent to the damaged surface (Cl-NERF pH sensor).
In control (TFF2+/+ and TFF2+/−) mice, damaged cells were exfoliated and the damaged epithelium was repaired by indomethacin. The resting surface pH was similar between control and TFF2-KO animals, but the post-LPD alkalisation of surface pH observed in control mice (ΔpH 0.3±0.05, n=21) was attenuated in the TFF2-KO stomach (ΔpH −0.08±0.09, n=18). Recobinant rat TFF3 partially rescued the attenuated surface pH change in TFF2-KO stomach, in the presence or absence of indomethacin.
In the gastric epithelium in vivo, TFFs promote epithelial restitution via a mechanism that does not require cyclooxygenase activation. A novel role for TFFs to affect gastric surface pH is observed.
Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). In the current studies we analyzed the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis.
Using a TNF-driven model of ileitis (i.e. TNFΔARE) that recapitulates many features of Crohn’s disease (CD), we assessed dynamic changes in the frequency and functional state of MP within the inflamed ileum by flow cytometry, immunofluorescence and real-time reverse-transcription polymerase chain reaction and by generating CX3CR1 GFP-reporter TNFΔARE mice. Finally, we assessed the effect of Flt3-L supplementation on the severity of ileitis, the frequency of CD103+ DC and of FoxP3+ Tregs in TNFΔARE mice.
CD11cHi/MHCII+ MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX3CR1+ MP subpopulation. This coincided with a decreased pro-regulatory CD103+ DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103+ DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4+/CD25+/FoxP3+ Tregs.
Our findings support a role for Flt3-L as a potential therapeutic in Crohn’s-like ileitis.
Inflammatory bowel disease; mucosal immunology; dendritic cells; small intestine
Trypsinogen activation is sufficient to induce acute pancreatitis in an experimental model. However, whether it is a requirement for the pathogenesis of acute and chronic pancreatitis remains to be explored.
Trypsin; Acute pancreatitis; Chronic pancreatitis
Background and Aims
Bariatric surgery is increasingly performed worldwide to treat morbid obesity and is also known as metabolic surgery to reflect its beneficial metabolic effects especially with respect to improvement in type 2 diabetes. Understanding surgical weight loss mechanisms and metabolic modulation is required to enhance patient benefits and operative outcomes.
We apply a parallel and statistically integrated metagenomic and metabonomic approach to characterize Roux-en-Y gastric bypass (RYGB) effects in a rat model.
We show substantial shifts of the main gut phyla towards higher levels of Proteobacteria (52-fold) specifically Enterobacter hormaechei. We also find low levels of Firmicutes (4.5-fold) and Bacteroidetes (2-fold) in comparison to sham-operated rats. Faecal extraction studies reveal a decrease in faecal bile acids and a shift from protein degradation to putrefaction through decreased faecal tyrosine with concomitant increases in faecal putrescine and diamnoethane. We find decreased urinary amines and cresols and demonstrate indices of modulated energy metabolism post-RYGB including decreased urinary succinate, 2-oxoglutarate, citrate and fumarate. These changes could also indicate renal tubular acidosis, which associates with increased flux of mitochondrial tricarboxylic acid cycle intermediates. A surgically-induced effect on the gut-brain-liver metabolic axis is inferred by increased neurotropic compounds; faecal γ-aminobutyric acid (GABA) and glutamate.
This profound co-dependence of mammalian and microbial metabolism, which is systematically altered following RYGB surgery, suggests that RYGB exerts local and global metabolic activities. The effect of RYGB surgery on the host metabolic-microbial crosstalk augments our understanding of the metabolic phenotype of bariatric procedures and can facilitate enhanced treatments for obesity-related diseases.
bariatric surgery; NMR spectroscopy; UPLC-MS; obesity; bile acid
It is postulated that high serum levels of insulin and insulin growth factor 1 (IGF-1) mediate obesity-associated carcinogenesis. The relationship of insulin, IGF-1 and IGF binding proteins (IGFBP) with Barrett’s oesophagus (BO) has not been well examined.
Serum levels of insulin and IGFBPs in patients with BO were compared with two separate control groups: subjects with gastro-oesophageal reflux disease (GORD) and screening colonoscopy controls. Fasting insulin, IGF-1 and IGFBPs were assayed in the serum of BO cases (n = 135), GORD (n = 135) and screening colonoscopy (n = 932) controls recruited prospectively at two academic hospitals. Logistic regression was used to estimate the risk of BO.
Patients in the highest tertile of serum insulin levels had an increased risk of BO compared with colonoscopy controls (adjusted OR 2.02, 95% CI 1.15 to 3.54) but not compared with GORD controls (adjusted OR 1.55, 95% CI 0.76 to 3.15). Serum IGF-1 levels in the highest tertile were associated with an increased risk of BO (adjusted OR 4.05, 95% CI 2.01 to 8.17) compared with the screening colonoscopy control group but were not significantly different from the GORD control group (adjusted OR 0.57, 95% CI 0.27 to 1.17). IGFBP-1 levels in the highest tertile were inversely associated with a risk of BO in comparison with the screening colonoscopy controls (adjusted OR 0.11, 95% CI 0.05 to 0.24) but were not significantly different from the GORD control group (adjusted OR 1.04, 95% CI 0.49 to 2.16). IGFBP-3 levels in the highest tertile were inversely associated with the risk of BO compared with the GORD controls (OR 0.36, 95% CI 0.16 to 0.81) and also when compared with the colonoscopy controls (OR 0.40, 95% CI 0.20 to 0.79).
These results provide support for the hypothesis that the insulin/IGF signalling pathways have a role in the development of BO.
colon cancer; rectal cancer; subsite; tumor location; personalised medicine
Colorectal cancer is typically classified into proximal colon, distal colon, and rectal cancer. Tumor genetic and epigenetic features differ by tumor location. Considering a possible role of bowel contents (including microbiome) in carcinogenesis, we hypothesized that tumor molecular features might gradually change along bowel subsites, rather than abruptly change at splenic flexure.
Utilizing 1443 colorectal cancers in two U.S. nationwide prospective cohort studies, we examined the frequencies of molecular features [CpG island methylator phenotype (CIMP), microsatellite instability (MSI), LINE-1 methylation, and BRAF, KRAS, and PIK3CA mutations] along bowel subsites (rectum, rectosigmoid junction, sigmoid, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon, and cecum). Linearity and non-linearity of molecular relations along subsites were statistically tested by multivariate logistic or linear regression analysis.
The frequencies of CIMP-high, MSI-high, and BRAF mutation gradually increased from rectum (<2.3%) to ascending colon (36–40%), followed by falls in the cecum (12–22%). By linearity tests, these molecular relations were significantly linear from rectum to ascending colon (p<0.0001), and there was little evidence for non-linearity (p>0.09). Cecal cancers exhibited the highest frequency of KRAS mutations (52% vs. 27–35% in other sites; p<0.0001).
The frequencies of CIMP-high, MSI-high, and BRAF mutation in cancer increased gradually along colorectum subsites from rectum to ascending colon. Our novel data challenge the common conception of discrete molecular features of proximal vs. distal colorectal cancers, and have substantial impact on clinical, translational, and epidemiology research, which has typically been performed with dichotomous classification of proximal vs. distal tumors.
colon cancer; epigenetics; genomic instability; continuum; RAS; RAF; PI3K
Background and aims
The authors’ goal was to measure pH at the gastric surface (pHo) to understand how acid secretion affects the repair of microscopic injury to the gastric epithelium.
Microscopic gastric damage was induced by laser light, during confocal/two-photon imaging of pH-sensitive dyes (Cl-NERF, BCECF) that were superfused over the mucosal surface of the exposed gastric corpus of anaesthetised mice. The progression of repair was measured in parallel with pHo. Experimental conditions included varying pH of luminal superfusates, and using omeprazole (60 mg/kg ip) or famotidine (30 mg/kg ip) to inhibit acid secretion.
Similar rates of epithelial repair and resting pHo values (~pH 4) were reported in the presence of luminal pH 3 or pH 5. Epithelial repair was unreliable at luminal pH 2 and pHo was lower (2.5±0.2, P <0.05 vs pH 3). Epithelial repair was slower at luminal pH 7 and pHo was higher (6.4±0.1, P<0.001). In all conditions, pHo increased adjacent to damage. At luminal pH 3 or pH 7, omeprazole reduced maximal damage size and accelerated epithelial repair, although only at pH 3 did omeprazole further increase surface pH above the level caused by imposed damage. At luminal pH 7, famotidine also reduced maximal damage size and accelerated epithelial repair. Neither famotidine nor omeprazole raised plasma gastrin levels during the time course of the experiments.
Epithelial repair in vivo is affected by luminal pH variation, but the beneficial effects of acutely blocking acid secretion extend beyond simply raising luminal and/or surface pH.
The death rate of mature hepatocytes is chronically increased in various liver diseases, triggering responses that prevent liver atrophy, but often cause fibrosis. Mice with targeted disruption of Inhibitor kappa beta kinase (Ikk) in hepatocytes (HEP) provide a model to investigate this process because inhibiting Ikk-NFκB signaling in hepatocytes increases their apoptosis.
Cell proliferation, apoptosis, progenitors, fibrosis, and production of Hedgehog (Hh) ligands (progenitor and myofibroblast growth factors) were compared in ΔHEP and control mice before and after feeding methionine choline-deficient ethionine-supplemented (MCDE) diets. Ikkβ was deleted from primary hepatocytes to determine effects on Hh ligand production; Hh signaling was inhibited directly in progenitors to determine effects on viability. Liver sections from patients were examined to assess relationships between hepatocyte production of Hh ligands, accumulation of myofibroblastic cells, and liver fibrosis.
Disrupting the Ikk-NFkB pathway in hepatocytes inhibited their proliferation but induced their production of Hh ligands. The latter provided viability signals for progenitors and myofibroblasts, enhancing accumulation of these cell types, and causing fibrogenesis. Findings in the mouse models were recapitulated in diseased human livers.
Dying mature hepatocytes produce Hh ligands which promote the compensatory outgrowth of progenitors and myofibroblasts. These results help to explain why diseases that chronically increase hepatocyte death promote cirrhosis.
Hedgehog; Progenitors; Ikkβ; nonalcoholic steatohepatitis (NASH)