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This 1-year study aimed to assess low-dose budesonide therapy for maintenance of clinical remission in patients with collagenous colitis.
A prospective, randomised, placebo-controlled study beginning with an 8-week open-label induction phase in which patients with histologically confirmed active collagenous colitis received budesonide (Budenofalk, 9 mg/day initially, tapered to 4.5 mg/day), after which 92 patients in clinical remission were randomised to budesonide (mean dose 4.5 mg/day; Budenofalk 3 mg capsules, two or one capsule on alternate days) or placebo in a 12-month double-blind phase with 6 months treatment-free follow-up. Primary endpoint was clinical remission throughout the double-blind phase.
Clinical remission during open-label treatment was achieved by 84.5% (93/110 patients). The median time to remission was 10.5 days (95% CI (9.0 to 14.0 days)). The maintenance of clinical remission at 1 year was achieved by 61.4% (27/44 patients) in the budesonide group versus 16.7% (8/48 patients) receiving placebo (treatment difference 44.5% in favour of budesonide; 95% CI (26.9% to 62.7%), p<0.001). Health-related quality of life was maintained during the 12-month double-blind phase in budesonide-treated patients. During treatment-free follow-up, 82.1% (23/28 patients) formerly receiving budesonide relapsed after study drug discontinuation. Low-dose budesonide over 1 year resulted in few suspected adverse drug reactions (7/44 patients), all non-serious.
Budesonide at a mean dose of 4.5 mg/day maintained clinical remission for at least 1 year in the majority of patients with collagenous colitis and preserved health-related quality of life without safety concerns. Treatment extension with low-dose budesonide beyond 1 year may be beneficial given the high relapse rate after budesonide discontinuation.
http://www.clinicaltrials.gov (NCT01278082) and
Fibroblast growth factors (Fgfs) are key orchestrators of development, and a role of Fgfs in tissue repair is emerging. Here we studied the consequences of inducible loss of Fgf receptor (Fgfr) 4, the major Fgf receptor (Fgfr) on hepatocytes, alone or in combination with Fgfr1 and Fgfr2, for liver regeneration after PH.
We used siRNA delivered via nanoparticles combined with liver-specific gene knockout to study Fgfr function in liver regeneration. Liver or blood samples were analysed using histology, immunohistochemistry, real-time RT-PCR, western blotting and ELISA.
siRNA-mediated knockdown of Fgfr4 severely affected liver regeneration due to impairment of hepatocyte proliferation combined with liver necrosis. Mechanistically, the proliferation defect resulted from inhibition of an Fgf15-Fgfr4-Stat3 signalling pathway, which is required for injury-induced expression of the Foxm1 transcription factor and subsequent cell cycle progression, while elevated levels of intrahepatic toxic bile acids were identified as the likely cause of the necrotic damage. Failure of liver mass restoration in Fgfr4 knockdown mice was prevented at least in part by compensatory hypertrophy of hepatocytes. Most importantly, our data revealed partially redundant functions of Fgf receptors in the liver, since knockdown of Fgfr4 in mice lacking Fgfr1 and Fgfr2 in hepatocytes caused liver failure after PH due to severe liver necrosis and a defect in regeneration.
These results demonstrate that Fgfr signalling in hepatocytes is essential for liver regeneration and suggest activation of Fgfr signalling as a promising approach for the improvement of the liver's regenerative capacity.
Oral methotrexate (MTX) administration avoids weekly injections, reduces costs and may improve quality of life of patients with Crohn's disease (CD), especially children. Routes of administration have never been systematically compared in CD. We aimed to compare effectiveness and safety of orally (PO) versus subcutaneously (SC) administered MTX in paediatric CD.
226 children with CD treated with oral or subcutaneous MTX were included in a multicentre, retrospective 1-year cohort study (62% boys, mean age 13.8±2.8 years, 88% previous thiopurines). 38 (17%) were initially commenced on oral, 98 (43%) started subcutaneous and switched to oral and 90 (40%) were treated with subcutaneous only. Matching and 'doubly robust' weighted regression models were based on the propensity score method, controlling for confounding-by-indication bias. 11/23 pretreatment variables were different between the groups, but the propensity score modelling successfully balanced the treatment groups.
76 children (34%) had sustained steroid-free remission with a difference that did not reach significance between the PO and the SC groups (weighted OR=1.72 (95% CI 0.5 to 5.9); p=0.52). There were no differences in need for treatment escalation (p=0.24), elevated liver enzymes (p=0.59) or nausea (p=0.85). Height velocity was lower in the PO group (p=0.006) and time to remission was delayed in the PO group (p=0.036; Fleming (0, 1) test).
In this largest paediatric CD cohort to date, SC administered MTX was superior to PO, but only in some of the outcomes and with a modest effect size. Therefore, it may be reasonable to consider switching children in complete remission treated with subcutaneous MTX to the oral route with close monitoring of inflammatory markers and growth.
IBD, which primarily includes UC and Crohn's disease (CD), is a progressive, chronic and relapsing condition. This debilitating disease is steadily becoming a worldwide medical concern, with increasing prevalence and incidence in both industrialised and developing countries.
Post-colonoscopy colorectal cancer (PCCRC) is a key quality indicator of colonoscopy. This study compares methods for defining PCCRC rates, proposes a new method of calculating them and quantifies them across the English National Health Service (NHS).
This retrospective observational population-based study involved all individuals with a first primary diagnosis of colorectal cancer made between 2001 and 2010 and treated in the English NHS. Previously published methods for deriving PCCRC rates were applied to the linked routine health data for this population to investigate the effect on the rate. A new method, based on the year of the colonoscopy rather than colorectal cancer diagnosis, was then used to calculate PCCRC rates.
Of 297 956 individuals diagnosed with colorectal cancer, a total of 94 648 underwent a colonoscopy in the 3 years prior to their diagnosis. The application of the published methods and exclusion criteria to the dataset produced significantly different PCCRC rates from 2.5% to 7.7%. The new method demonstrates that PCCRC rates within 3 years of colonoscopy (without exclusions) decreased in the English NHS over 8 years, falling from 10.6% to 7.3% for colonoscopies performed in 2001 and 2007 respectively.
The method used to determine PCCRC rates significantly affects findings with potential to substantially underestimate rates. To enable international benchmarking there needs to be a standardised method for defining PCCRC. This study proposes a new methodology using colonoscopy as a denominator and between 2001 and 2007 this method indicated an 8.6% PCCRC rate across the English NHS. It also demonstrated PCCRC rates have fallen over time.
Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function.
Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220 days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation.
No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30 days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220 days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220 days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats.
BB-DP rats of 220 days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.
However, in hitherto conducted studies in Asian and Western patients with HBeAg-negative chronic hepatitis B, virological relapses alone defined as HBV DNA >2000 IU/mL with normal ALT levels, have first been monitored at frequent intervals while initiation of NA therapy again was considered only if ALT levels also increased >2x upper limit of normal, that means when virological relapses changed to clinical ones.
Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects.
To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients.
We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition.
Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours.
Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.
Whether there is distinct pathogenesis in subgroups of functional dyspepsia (FD), the postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS) remains controversial. We aimed to identify the risk factors of FD and its subgroups in the Chinese population.
Patients with dyspepsia and healthy subjects who underwent gastric cancer screening were enrolled in this multicentre study from 2010 to 2012. All patients were evaluated by questionnaire, oesophagoduodenoscopy, histological examination and Helicobacter pylori tests. Subgroups of FD were classified according to the Rome III criteria. Psychiatric stress was assessed by the short form Brief Symptom Rating Scale. CagA and VacA genotypes were determined by PCR.
Of 2378 patients screened for eligibility, 771 and 491 fulfilled the diagnostic criteria of uninvestigated dyspepsia and FD, respectively. 298 (60.7%) and 353 (71.9%) individuals were diagnosed with EPS and PDS, respectively, whereas 169 (34.4%) had the overlap syndrome. As compared with 1031 healthy controls, PDS and EPS shared some common risk factors, including younger age (OR 0.95; 99.5% CI 0.93 to 0.98), non-steroidal anti-inflammatory drugs (OR 6.60; 99.5% CI 3.13 to 13.90), anxiety (OR 3.41; 99.5% CI 2.01 to 5.77) and concomitant IBS (OR 6.89; 99.5% CI 3.41 to 13.94). By contrast, H. pylori (OR 1.86; 99.5% CI 1.01 to 3.45), unmarried status (OR 4.22; 99.5% CI 2.02 to 8.81), sleep disturbance (OR 2.56; 99.5% CI 1.29 to 5.07) and depression (OR 2.34; 99.5% CI 1.04 to 5.36) were associated with PDS. Moderate to severe antral atrophy and CagA positive strains were also more prevalent in PDS.
Different risk factors exist among FD subgroups based on the Rome III criteria, indicating distinct aetiopathogenesis of the subdivisions that may necessitate different therapeutic strategies.
Although serrated polyps may be precursors of colorectal cancer (CRC), prospective data on the long-term CRC risk in individuals with serrated polyps are lacking.
In a population-based randomised trial, 12 955 individuals aged 50–64 years were screened with flexible sigmoidoscopy, while 78 220 individuals comprised the control arm. We used Cox models to estimate HRs with 95% CIs for CRC among individuals with ≥1 large serrated polyp (≥10 mm in diameter), compared with individuals with adenomas at screening, and to population controls, and multivariate logistic regression to assess polyp risk factors for CRC.
A total of 103 individuals had large serrated polyps, of which 81 were included in the analyses. Non-advanced adenomas were found in 1488 individuals, advanced adenomas in 701. Median follow-up was 10.9 years. Compared with the control arm, the HR for CRC was 2.5 (95% CI 0.8 to 7.8) in individuals with large serrated polyps, 2.0 (95% CI 1.3 to 2.9) in individuals with advanced adenomas and 0.6 (95% CI 0.4 to 1.1) in individuals with non-advanced adenomas. A large serrated polyp was an independent risk factor for CRC, adjusted for histology, size and multiplicity of concomitant adenomas (OR 3.3; 95% CI 1.3 to 8.6). Twenty-three large serrated polyps found at screening were left in situ for a median of 11.0 years. None developed into a malignant tumour.
Individuals with large serrated polyps have an increased risk of CRC, comparable with individuals with advanced adenomas. However, this risk may not be related to malignant growth of the serrated polyp.
The Norwegian Colorectal Cancer Screening trial is registered at clinicaltrials.gov (NCT00119912).
What is the diagnosis?
EUS revealed multiple well-defined hypoechoic 1.5–2 cm lesions within the gastric wall arising from muscularis propria...]]>
STK33 has been reported to play an important role in cancer cell proliferation. We investigated the role of STK33 in hepatocellular carcinoma (HCC) and its underlying mechanisms.
251 patients with HCC were analysed for association between STK33 expression and clinical stage and survival rate. Tamoxifen (TAM)-inducible, hepatocyte-specific STK33 transgenic and knockout mice models were used to study the role of STK33 in liver tumorigenesis. HCC cell lines were used to study the role of STK33 in cell proliferation in vitro and in vivo.
STK33 expression was found to be frequently upregulated in patients with HCC. Significant associations were found between increased expression of STK33 and advanced HCC staging and shorter disease-free survival of patients. Overexpression of STK33 increased HCC cell proliferation both in vitro and in vivo, whereas suppression of STK33 inhibited this effect. Using a TAM-inducible, hepatocyte-specific STK33 transgenic mouse model, we found that overexpression of STK33 resulted in increased hepatocyte proliferation, leading to tumour cell burst. Using a TAM-inducible, hepatocyte-specific STK33 knockout mouse model, we found that, when subjected to the diethylnitrosamine (DEN) liver cancer bioassay, STK33KOflox/flox, Alb-ERT2-Cre mice exhibited a markedly lower incidence of tumour formation compared with control mice. The underlying mechanism may be that STK33 binds directly to c-Myc and increases its transcriptional activity. In particular, the C-terminus of STK33 blocks STK33/c-Myc association, downregulates HCC cell proliferation, and reduces DEN-induced liver tumour cell number and tumour size.
STK33 plays an essential role in hepatocellular proliferation and liver tumorigenesis. The C-terminus of STK33 could be a potential therapeutic target in the treatment of patients with STK33-overexpressed HCC.
The VIRGIN Study was a multicentre, blinded, prospective, analytical trial carried out at...]]>
To elucidate the association between antiviral therapy and extrahepatic outcomes in individuals infected with HCV.
This nationwide cohort study screened 293 480 Taiwanese residents with HCV infection and excluded those with substantial comorbidity. A total of 12 384 eligible patients who had received pegylated interferon plus ribavirin between 1 October 2003 and 31 December 2010 were enrolled in the treated cohort; they were matched 1 : 2 with 24 768 untreated controls in the propensity score and post-diagnosis treatment-free period. The incidences of end-stage renal disease (ESRD), acute coronary syndrome (ACS), ischaemic stroke and catastrophic autoimmune diseases were calculated after adjustment for competing mortality.
The treated and untreated cohorts were followed up for a mean (±SD) duration of 3.3 (±2.5) and 3.2 (±2.4) years, respectively, until 31 December 2011. The calculated 8-year cumulative incidences of ESRD, ACS, ischaemic stroke and autoimmune catastrophes between treated and untreated patients were 0.15% vs 1.32% (p<0.001), 2.21% vs 2.96% (p=0.027), 1.31% vs 1.76% (p=0.001) and 0.57% vs 0.49% (p=0.816), respectively. Multivariate-adjusted Cox regression revealed that antiviral treatment was associated with lower risks of ESRD (HR 0.15; 95% CI 0.07 to 0.31; p<0.001), ACS (HR 0.77; 95% CI 0.62 to 0.97; p=0.026) and ischaemic stroke (HR 0.62; 95% CI 0.46 to 0.83; p=0.001), but unrelated to autoimmune catastrophes. These favourable associations were invalid in incompletely treated patients with duration <16 weeks.
Antiviral treatment for HCV is associated with improved renal and circulatory outcomes, but unrelated to catastrophic autoimmune diseases.
NSBBs have been studied since the early 1960s and have been a cornerstone in clinical hepatology where they have benefitted more patients and saved more lives than any other drug in this field over the past three decades (
In 2010, the scientific community was shocked as data came out suggesting that NSBBs were...]]>
Diminutive (≤5 mm) colorectal polyps are common, and overwhelmingly benign. Routinely, after polypectomy, they are examined pathologically to determine the surveillance intervals. Advances in equipment and techniques, such as narrow-band imaging (NBI) colonoscopy, now permit reliable real-time optical diagnosis.
We conducted a randomised single-masked study involving three institutions to determine whether optical diagnosis of diminutive colorectal polyps meets clinical practice standards and reduces the need for histopathology. We randomly assigned eligible patients undergoing routine high-definition colonoscopy to optical diagnosis using near focus versus standard view, using computer-generated block sequence. By validated criteria, we rendered an optical diagnosis and a confidence level (high vs low) for all polyps, using NBI. Our primary endpoint was the number of accurate high-confidence optical diagnoses compared with central blinded pathology in the two groups. We analysed data using intention to treat.
We enrolled 558 subjects, and randomly assigned 281 to near focus and 277 to standard view optical diagnosis. We detected 1309 predominantly diminutive (74.5%) and neoplastic (60.0%) polyps. Endoscopists were significantly more likely, OR 2.2 (95% CI 1.6 to 3.0, p<0.0001), to make a high-confidence optical diagnosis with near focus (85.1%) than standard (72.6%) view. High-confidence diagnoses had 96.4% and 92.0% negative predictive value, respectively. Of all polyps, 75.3% (95% CI71.3% to 78.9%) had a high-confidence accurate prediction using near focus, compared with 63.1% (95% CI 58.5% to 67.6%) using standard view. Optical versus histopathological diagnosis showed excellent agreement between the surveillance intervals, 93.5% in near focus and 92.2% in standard view. The median diagnosis time was 14 s.
Real-time optical diagnosis using NBI colonoscopy may replace the pathology diagnosis for the majority of diminutive colorectal polyps. Using colonoscopy with near focus view increases the confidence level of the optical diagnosis. Optical diagnosis would be a paradigm shift in clinical practice of colonoscopy for colorectal cancer screening.
ClinicalTrials.gov Identifier: NCT01288833.
Differences in gastric cancer (GC) clinical outcomes between patients in Asian and non-Asian countries has been historically attributed to variability in clinical management. However, recent international Phase III trials suggest that even with standardised treatments, GC outcomes differ by geography. Here, we investigated gene expression differences between Asian and non-Asian GCs, and if these molecular differences might influence clinical outcome.
We compared gene expression profiles of 1016 GCs from six Asian and three non-Asian GC cohorts, using a two-stage meta-analysis design and a novel biostatistical method (RUV-4) to adjust for technical variation between cohorts. We further validated our findings by computerised immunohistochemical analysis on two independent tissue microarray (TMA) cohorts from Asian and non-Asian localities (n=665).
Gene signatures differentially expressed between Asians and non-Asian GCs were related to immune function and inflammation. Non-Asian GCs were significantly enriched in signatures related to T-cell biology, including CTLA-4 signalling. Similarly, in the TMA cohorts, non-Asian GCs showed significantly higher expression of T-cell markers (CD3, CD45R0, CD8) and lower expression of the immunosuppressive T-regulatory cell marker FOXP3 compared to Asian GCs (p<0.05). Inflammatory cell markers CD66b and CD68 also exhibited significant cohort differences (p<0.05). Exploratory analyses revealed a significant relationship between tumour immunity factors, geographic locality-specific prognosis, and postchemotherapy outcomes.
Analyses of >1600 GCs suggest that Asian and non-Asian GCs exhibit distinct tumour immunity signatures related to T-cell function. These differences may influence geographical differences in clinical outcome, and the design of future trials particularly in immuno-oncology.
The performance of the NIHR Clinical Research Network Portfolio...]]>
Epigenetic mechanisms are potential targets to relieve somatic pain. However, little is known whether epigenetic regulation interferes with visceral pain. Previous studies show that oestrogen facilitates visceral pain. This study aimed to determine whether histone hyperacetylation in the spinal cord could attenuate oestrogen-facilitated visceral pain.
The effect of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) on the magnitude of the visceromotor response (VMR) to colorectal distention was examined in ovariectomised rats with/without oestrogen replacement. An additional interaction with the metabotropic glutamate receptor 2/3 (mGluR2/3) antagonist LY341495 was tested. The levels of acetylated histone and mGluR2 mRNA and protein were analysed. The binding of acetylated H3 and oestrogen receptor α (ERα) to the GRM2 promoter was measured by chromatin immunoprecipitation coupled with qPCR.
In ovariectomised rats, 17β-estradiol (E2), but not safflower oil, increased the magnitude of the VMR to colorectal distention. SAHA attenuated the E2-facilitated VMR, but had no effect in safflower oil-treated rats. Subsequent spinal administration of LY341495 reversed the antinociceptive effect of SAHA in E2 rats. In addition, SAHA increased mGluR2 mRNA and protein in the spinal dorsal horn following E2, but not vehicle, treatment. In contrast, neither E2 nor SAHA alone altered mGluR2 mRNA. SAHA increased binding of H3K9ac and ERα to the same regions of the GRM2 promoter in E2-SAHA-treated animals.
Histone hyperacetylation in the spinal cord attenuates the pronociceptive effects of oestrogen on visceral sensitivity, suggesting that epigenetic regulation may be a potential approach to relieve visceral pain.
The expense of new therapies for HCV infection may force health systems to prioritise the treatment of certain patient groups over others. Our objective was to forecast the population impact of possible prioritisation strategies for the resource-rich setting of Scotland.
We created a dynamic Markov simulation model to reflect the HCV-infected population in Scotland. We determined trends in key outcomes (e.g. incident cases of chronic infection and severe liver morbidity (SLM)) until the year 2030, according to treatment strategies involving prioritising, either: (A) persons with moderate/advanced fibrosis or (B) persons who inject drugs (PWID).
Continuing to treat the same number of patients with the same characteristics will give rise to a fall in incident infection (from 600 cases in 2015 to 440 in 2030) and a fall in SLM (from 195 cases in 2015 to 145 in 2030). Doubling treatment-uptake and prioritising PWID will reduce incident infection to negligible levels (<50 cases per year) by 2025, while SLM will stabilise (at 70–75 cases per year) in 2028. Alternatively, doubling the number of patients treated, but, instead, prioritising persons with moderate/advanced fibrosis will reduce incident infection less favourably (only to 280 cases in 2030), but SLM will stabilise by 2023 (i.e. earlier than any competing strategy).
Prioritising treatment uptake among PWID will substantially impact incident transmission, however, this approach foregoes the optimal impact on SLM. Conversely, targeting those with moderate/advanced fibrosis has the greatest impact on SLM but is suboptimal in terms of averting incident infection.
IBD is a group of complex, systemic disorders associated with intestinal inflammation and extraintestinal manifestations. Recent studies revealed Mendelian forms of IBD, which contributed significantly to our understanding of disease pathogenesis and the heritability of IBD.
We performed exome sequencing in a family with Crohn's disease (CD) and severe autoimmunity, analysed immune cell phenotype and function in affected and non-affected individuals, and performed in silico and in vitro analyses of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) structure and function.
A novel missense variant was identified in CTLA4 encoding CTLA-4, a coinhibitory protein expressed by T cells and required for regulation of T cell activation. The residue affected by the mutation, CTLA-4 Tyr60, is evolutionarily highly conserved, and the identified Y60C variant is predicted to affect protein folding and structural stability and demonstrated to cause impaired CTLA-4 dimerisation and CD80 binding. Intestinal inflammation and autoimmunity in carriers of CTLA-4 Y60C exhibit incomplete penetrance with a spectrum of clinical presentations ranging from asymptomatic carrier status to fatal autoimmunity and intestinal inflammation. In a clinically affected CTLA-4 Y60C carrier, T cell proliferation was increased in vitro and associated with an increased ratio of memory to naive T cells in vivo, consistent with impaired regulation of T cell activation.
Our results support the concept that variants in CTLA4 provide the basis for a novel Mendelian form of early-onset CD associated with systemic autoimmunity. Incomplete penetrance of autoimmunity further indicates the presence of other genetic and/or environmental modifiers.
Histopathology is potentially an important outcome measure in UC. Multiple histological disease activity (HA) indices, including the Geboes score (GS) and modified Riley score (MRS), have been developed; however, the operating properties of these instruments are not clearly defined. We assessed the reproducibility of existing measures of HA.
Five experienced pathologists with GI pathology fellowship training and expertise in IBD evaluated, on three separate occasions at least two weeks apart, 49 UC colon biopsies and scored the GS, MRS and a global rating of histological severity using a 100 mm visual analogue scale (VAS). The reproducibility of each grading system and for individual instrument items was quantified by estimates of intraclass correlation coefficients (ICCs) based on two-way random effects models. Uncertainty of estimates was quantified by 95% two-sided CIs obtained using the non-parametric cluster bootstrap method. Biopsies responsible for the greatest disagreement based on the ICC estimates were identified. A consensus process was used to determine the most common sources of measurement disagreement. Recommendations for minimising disagreement were subsequently generated.
Intrarater ICCs (95% CIs) for the total GS, MRS and VAS scores were 0.82 (0.73 to 0.88), 0.71 (0.63 to 0.80) and 0.79 (0.72 to 0.85), respectively. Corresponding inter-rater ICCs were substantially lower: 0.56 (0.39 to 0.67), 0.48 (0.35 to 0.66) and 0.61 (0.47 to 0.72). Correlation between the GS and VAS was 0.62 and between the MRS and VAS was 0.61.
Although 'substantial' to 'almost perfect' ICCs for intrarater agreement were found in the assessment of HA in UC, ICCs for inter-rater agreement were considerably lower. According to the consensus process results, standardisation of item definitions and modification of the existing indices is required to create an optimal UC histological instrument.
An 81-year-old man underwent percutaneous mitral valve repair (MitraClip) due to severe symptomatic mitral valve regurgitation. Coronary heart disease was excluded prior to mitral valve repair. Approximately 8 h after the uneventful and successful intervention, the patient presented with progressive abdominal pain, in particular tenderness in the right upper quadrant with guarding. Laboratory tests did not reveal any acute alterations; serum lactate was 2.4 mmol/L. Bedside ultrasonography showed a distended ascending colon with a pronounced hyperechoic large bowel wall (
What is the correct imaging diagnosis and the underlying condition?
Ultrasonography and CT imaging revealed pneumatosis intestinalis (PI) of the ascending colon (
Several genetic risk factors have been identified for non-alcoholic chronic pancreatitis (NACP). A genome-wide association study reported an association of chronic pancreatitis (CP) with variants in PRSS1–PRSS2 (rs10273639; near the gene encoding cationic trypsinogen) and CLDN2–MORC4 loci (rs7057398 in RIPPLY1 and rs12688220 in MORC4). We aimed to refine these findings in a large European cohort.
We studied 3062 patients with alcohol-related CP (ACP) or NACP and 5107 controls. Also, 1559 German patients with alcohol-associated cirrhosis or alcohol dependence were included for comparison. We performed several meta-analyses to examine genotype–phenotype relationships.
Association with ACP was found for rs10273639 (OR, 0.63; 95% CI 0.55 to 0.72). ACP was also associated with variants rs7057398 and rs12688220 in men (OR, 2.26; 95% CI 1.94 to 2.63 and OR, 2.66; 95% CI 2.21 to 3.21, respectively) and in women (OR, 1.57; 95% CI 1.14 to 2.18 and OR 1.71; 95% CI 1.41 to 2.07, respectively). Similar results were obtained when German patients with ACP were compared with those with alcohol-associated cirrhosis or alcohol dependence. In the overall population of patients with NACP, association with rs10273639 was absent (OR, 0.93; 95% CI 0.79 to 1.01), whereas rs7057398 of the X chromosomal single nucleotide polymorphisms was associated with NACP in women only (OR, 1.32; 95% CI 1.15 to 1.51).
The single-nucleotide polymorphisms rs10273639 at the PRSS1–PRSS2 locus and rs7057398 and rs12688220 at the CLDN2–MORC4 locus are associated with CP and strongly associate with ACP, but only rs7057398 with NACP in female patients.
Quantifying excess cause-specific mortality among people with coeliac disease (CD) compared with the general population accounting for competing risks will allow accurate information to be given on risk of death from specific causes.
We identified from the Clinical Practice Research Datalink all patients with CD linked to Office for National Statistics between 1998 and 2012. We selected controls by frequency matching from the registered general practice population within 10-year age bands. We calculated the adjusted cumulative incidence (including adjustment for competing risks) and excess cumulative incidence for different causes of death up to 10 years from diagnosis.
Of the 10 825 patients with CD, 773 died within the study period. The overall mortality rate among patients with CD was 128/10 000 person years compared with 153/10 000 in controls (HR=0.94 95% CI 0.84 to 1.01). We found no overall difference in the cumulative incidence of respiratory disease, digestive disease or cancer related death among cases and controls. The adjusted cumulative incidence of death from cardiovascular deaths was slightly lower compared with those without CD diagnosis (CD 0.32% vs controls 0.41%) with a corresponding excess cumulative incidence of –0.08% (95% CI –0.13 to –0.04). However, patients with CD had 0.15% excess risk (95% CI 0.03 to 0.27) of deaths from non-Hodgkin's lymphoma from the general population baseline risk.
Overall, people with CD have no major excess risk of cancer, digestive disease or respiratory disease related or cardiovascular mortality compared with the general population. These findings should be reassuring to patients with CD and clinicians managing their care.
Shigeyasu et al
Shigeyasu et al
A 62-year-old woman presented with abdominal pain and diarrhoea. She had a history of primary biliary cirrhosis and renal transplantation secondary to reflux nephropathy. Cross-sectional imaging and a colonoscopy demonstrated terminal ileal inflammation and ulceration, but histological biopsies were inconclusive and cytomegalovirus staining was negative. The patient subsequently developed spontaneous small bowel perforation due to deep ulceration, necessitating an emergency right hemicolectomy. Her postoperative period was complicated by several episodes of rectal bleeding. After a prolonged admission at her local hospital, the patient was referred to our unit for further management.
On arrival, the patient's immunosuppression consisted of tacrolimus and low-dose prednisolone. Mycophenolate mofetil (MMF) had been discontinued prior to transfer due to concerns that MMF toxicity was the cause of her ileal disease. Laboratory evaluation revealed a haemoglobin of 83 g/L with a raised C-reactive protein (21 mg/L). CT angiography showed no active bleeding despite clinical evidence of ongoing rectal...]]>
Although low infliximab trough concentrations and antibodies to infliximab (ATI) are associated with poor outcomes in patients with Crohn's disease (CD), the clinical relevance of ATI in patients with adequate infliximab concentrations is uncertain. We evaluated this question using an assay sensitive for identification of ATI in the presence of infliximab.
In an observational study, 1487 trough serum samples from 483 patients with CD who participated in four clinical studies of maintenance infliximab therapy were analysed using a fluid phase mobility shift assay. Infliximab and ATI concentrations most discriminant for remission, defined as a C-reactive protein concentration of ≤5 mg/L, were determined by receiver operating characteristic curves. A multivariable regression model evaluated these factors as independent predictors of remission.
Based upon analysis of 1487 samples, 77.1% of patients had detectable and 22.9% had undetectable infliximab concentrations, of which 9.5% and 71.8%, respectively, were positive for ATI. An infliximab concentration of >2.79 μg/mL (area under the curve (AUC)=0.681; 95% CI 0.632 to 0.731) and ATI concentration of <3.15 U/mL (AUC=0.632; 95% CI 0.589 to 0.676) were associated with remission. Multivariable analysis showed that concentrations of both infliximab trough (OR 1.8; 95% CI 1.3 to 2.5; p<0.001) and ATI (OR 0.57; 95% CI 0.39 to 0.81; p=0.002) were independent predictors of remission.
The development of ATI increases the probability of active disease even at low concentrations and in the presence of a therapeutic concentration of drug during infliximab maintenance therapy. Evaluation of strategies to prevent ATI formation, including therapeutic drug monitoring with selective infliximab dose intensification, is needed.
The transcription factor SOX9 was recently shown to stimulate ductal gene expression in pancreatic acinar-to-ductal metaplasia and to accelerate development of premalignant lesions preceding pancreatic ductal adenocarcinoma (PDAC). Here, we investigate how SOX9 operates in pancreatic tumourigenesis.
We analysed genomic and transcriptomic data from surgically resected PDAC and extended the expression analysis to xenografts from PDAC samples and to PDAC cell lines. SOX9 expression was manipulated in human cell lines and mouse models developing PDAC.
We found genetic aberrations in the SOX9 gene in about 15% of patient tumours. Most PDAC samples strongly express SOX9 protein, and SOX9 levels are higher in classical PDAC. This tumour subtype is associated with better patient outcome, and cell lines of this subtype respond to therapy targeting epidermal growth factor receptor (EGFR/ERBB1) signalling, a pathway essential for pancreatic tumourigenesis. In human PDAC, high expression of SOX9 correlates with expression of genes belonging to the ERBB pathway. In particular, ERBB2 expression in PDAC cell lines is stimulated by SOX9. Inactivating Sox9 expression in mice confirmed its role in PDAC initiation; it demonstrated that Sox9 stimulates expression of several members of the ERBB pathway and is required for ERBB signalling activity.
By integrating data from patient samples and mouse models, we found that SOX9 regulates the ERBB pathway throughout pancreatic tumourigenesis. Our work opens perspectives for therapy targeting tumourigenic mechanisms.
The two major histological types of oesophageal cancer—adenocarcinoma (AC) and squamous cell carcinoma (SCC)—are known to differ greatly in terms of risk factors and epidemiology. To date, global incidence estimates for individual subtypes are still lacking. This study for the first time quantified the global burden of oesophageal cancer by histological subtype.
Where available, data from Cancer Incidence in Five Continents Vol. X (CI5X) were used to compute, age-specific, sex-specific and country-specific proportions of AC and SCC. Nine regional averages were computed for countries without CI5X data. The proportions were then applied to all oesophageal cancer cases from GLOBOCAN 2012 and age-standardised incidence rates calculated for both histological types.
Worldwide, an estimated 398 000 SCCs and 52 000 ACs of the oesophagus occurred in 2012, translating to incidence rates of 5.2 and 0.7 per 100 000, respectively. Although SCCs were most common in South-Eastern and Central Asia (79% of the total global SCC cases), the highest burden of AC was found in Northern and Western Europe, Northern America and Oceania (46% of the total global AC cases). Men had substantially higher incidence than women, especially in the case of AC (male to female ratio AC: 4.4; SCC: 2.7).
These first global estimates of oesophageal cancer incidence by histology suggested a high concentration of AC in high-income countries with men being at much greater risk. This quantification of incidence will aid health policy makers to plan appropriate cancer control measures in the future.
A 63-year-old woman was admitted to our gastroenterology unit to undergo a screening colonoscopy, scheduled for faecal occult blood test positivity. During the procedure, a flat, rectal granular lesion was found. With the use of magnification and narrow band image (NBI) technology, the lesion was diagnosed as a laterally spreading tumour (LST), granular mixed type, 30 mm in maximum diameter (
Prognostic stratification of patients with cirrhosis is common clinical practice. This study compares the prognostic accuracy (28-day and 90-day transplant-free mortality) of the acute-on-chronic liver failure (ACLF) classification (no ACLF, ACLF grades 1, 2 and 3) with that of acute kidney injury (AKI) classification (no AKI, AKI stages 1, 2 and 3).
The study was performed in 510 patients with an acute decompensation of cirrhosis previously included in the European Association for the Study of the Liver–Chronic Liver Failure consortium CANONIC study. ACLF was evaluated at enrolment and 48 h after enrolment, and AKI was evaluated at 48 h according to Acute Kidney Injury Network criteria.
240 patients (47.1%) met the criteria of ACLF at enrolment, while 98 patients (19.2%) developed AKI. The presence of ACLF and AKI was strongly associated with mortality. 28-day transplant-free mortality and 90-day transplant-free mortality of patients with ACLF (32% and 49.8%, respectively) were significantly higher with respect to those of patients without ACLF (6.2% and 16.4%, respectively; both p<0.001). Corresponding values in patients with and without AKI were 46% and 59%, and 12% and 25.6%, respectively (p<0.0001 for both). ACLF classification was more accurate than AKI classification in predicting 90-day mortality (area under the receiving operating characteristic curve=0.72 vs 0.62; p<0.0001) in the whole series of patients. Moreover, assessment of ACLF classification at 48 h had significantly better prognostic accuracy compared with that of both AKI classification and ACLF classification at enrolment.
ACLF stratification is more accurate than AKI stratification in the prediction of short-term mortality in patients with acute decompensation of cirrhosis.
A new scenario of therapy for chronic hepatitis C (CHC) is being established with the approval of sofosbuvir (SOF).
To estimate the cost-effectiveness of SOF-based regimens approved in the Summary of Product Characteristics (SmPC) versus the standard of care for different genotypes and patient populations (naive or pretreated).
A Markov model simulating CHC progression was used to estimate disease treatment costs and effects over patients' lifetimes, from the Spanish National Public Healthcare System perspective. Different therapeutic options were analysed for genotypes 1, 2 and 3 in naive population and for genotype 2 and 3 pretreated patients, according to data obtained from clinical trials. A one-way sensitivity analysis was performed to evaluate the uncertainty of certain parameters: treatment starting age, transition probabilities, drug costs and discount rate. A probabilistic sensitivity analysis was also carried out.
For the naive population, the option SOF+pegylated-interferon-α (pIFN)+ribavirin (RBV) for 12 weeks recorded in SmPC for genotype 1 and 3 versus pIFN+RBV for 24 weeks estimated an incremental cost-effectiveness ratio (ICER) below the 40 000/quality-adjusted life-year (QALY) benchmark. For the pretreated population, SOF triple therapy reached an ICER on the threshold limit for genotype 3. Other options included in SmPC for different genotypes exceeded the accepted efficiency limit in our setting.
The options that included SOF+RBV+pIFN in a 12-week course regimen fell below the efficiency threshold considered in our setting. IFN-free regimens administered for 24 weeks reached figures over the benchmark of 40 000/QALY.
Interleukin-13 (IL-13) has been implicated as a key driver of UC. This trial evaluates the efficacy and safety of tralokinumab, an IL-13-neutralising antibody, as add-on therapy in adults with moderate-to-severe UC despite standard treatments.
Non-hospitalised adults with UC (total Mayo score ≥6) were randomised to receive tralokinumab 300 mg or placebo subcutaneously every 2 weeks for 12 weeks. The primary end point was the rate of clinical response at week 8. Secondary efficacy end points included clinical remission and mucosal healing rates at week 8 and changes in total Mayo score, total modified Riley score, partial Mayo score and disease activity markers.
Clinical response rate was 38% (21/56) for tralokinumab vs 33% (18/55) for placebo (p=0.406). Clinical remission rate was 18% (10/56) vs 6% (3/55) (p=0.033) and mucosal healing rate was 32% (18/56) vs 20% (11/55) (p=0.104) for tralokinumab vs placebo. Changes to week 8 in total Mayo score and total modified Riley score were similar for tralokinumab and placebo (least-squares mean difference between groups: –0.49 (p=0.394) and 0.25 (p=0.449), respectively). Partial Mayo score at week 4 was lower with tralokinumab than placebo (least-squares mean difference between groups: –0.90 (p=0.041)). No consistent patterns were observed for disease activity markers. Tralokinumab had an acceptable safety profile.
Add-on therapy with tralokinumab did not significantly improve clinical response. However, the higher clinical remission rate with tralokinumab than placebo suggests that tralokinumab may benefit some patients with UC. Tralokinumab was well tolerated.
ClinicalTrials.gov number: NCT01482884.
To clarify the effectiveness of second-look endoscopy (SLE) at preventing bleeding after gastric endoscopic submucosal dissection (ESD).
A multicentre prospective randomised controlled non-inferiority trial was conducted at five referral institutions across Japan. Patients with a solitary gastric neoplasm were enrolled. Exclusion criteria were previous oesophagogastric surgery or radiation therapy; perforation and the administration of antithrombotics, steroids or non-steroidal anti-inflammatory drugs. Patients were assigned to the SLE group or the non-SLE group by a computer-generated random sequence after ESD and were treated perioperatively with a proton pump inhibitor. SLE was performed one day after ESD. The primary endpoint was post-ESD bleeding, defined as an endoscopically proven haemorrhage. The trial had the power to detect a non-inferiority criterion of 7% between the groups.
From February 2012 to February 2013, 130 and 132 patients were assigned to the SLE and the non-SLE groups, respectively. All patients were included in the intention-to-treat analysis of the primary endpoint. Post-ESD bleeding occurred in seven patients with (5.4%) SLE and five patients with (3.8%) non-SLE (risk difference –1.6% (95% CI –6.7 to 3.5); pnon-inferiority<0.001), meeting the non-inferiority criterion. All 12 patients with post-ESD bleeding and one patient with a delayed perforation were successfully managed with conservative treatment.
SLE after gastric ESD is not routinely recommended because it does not contribute to the prevention of post-ESD bleeding for patients with an average bleeding risk.
Increasing evidence suggests that genetic factors play a role in the multifactorial aetiology of liver fibrosis. In their recent paper, Zhao et al
Other studies have now confirmed earlier work showing that using an appropriate cut-off for IgA-class anti-tissue transglutaminase antibody (IgA-tTG), the diagnosis can be made with a positive predictive value (PPV) of 100% in a high proportion of adults with CD
Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4+ T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4+ T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action.
CD4+ T cells were studied in IL10–/– mice and Rag1–/– mice adoptively transferred with naive CD4+CD45RBhigh T cells, representing two well-characterised animal models of IBD and in patients with Crohn's disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors.
In mice, we identified bone marrow (BM) CD4+ T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α+ cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α+ cells was also detected in patients with Crohn's disease.
Our results highlight the osteoclastogenic function of the Th17 TNF-α+ cells that contribute to bone loss in vivo in IBD.
Consistent with the Xiao et al study, we have reported that blocking the HCV entry factor Neimann-Pick-C1-like-1 (NPC1L1) with the Food and Drug Administration (FDA)-approved drug ezetimibe synergistically inhibits chronic HCV infection in vitro when combined with interferon
Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD.
miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells.
The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon- inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses.
Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.
Laquinimod is an oral therapeutic agent under investigation for the treatment of Crohn's disease (CD), Huntington's disease, lupus nephritis and multiple sclerosis. This dose escalation study evaluated the safety and efficacy of laquinimod as induction therapy in patients with active moderate–severe CD.
Multicentre, double-blind, sequential-cohort, randomised controlled trial with laquinimod doses of 0.5, 1, 1.5 or 2 mg/day or placebo (n=45 per cohort randomised in a 2:1 ratio) for 8 weeks with 4-week follow-up. Stable concomittant therapies and prior use of anti-tumour necrosis factor agents were permitted. Comprehensive safety assessments were performed and efficacy analyses included the proportions of patients in clinical remission (CD Activity Index (CDAI) <150 and no treatment failure (TF)), and with a clinical response (70 or 100 point CDAI reduction from baseline or remission and no TF).
117 patients received laquinimod and 63 patients received placebo. The overall incidence of adverse events (AEs) in the laquinimod group was similar to the pooled placebo group (86.2%–96.7% vs 82.5%) and most AEs were mild to moderate in severity. Treatment with laquinimod 0.5 mg showed consistent effects on remission (48.3% (CI 31% to 66%) vs 15.9% (CI 9% to 27%)), response 100 (55.2% (CI 37% to 71%) vs 31.7% (CI 22% to 44%)) and response 70 (62.1% (CI 44% to 77%) vs 34.9% (CI 24% to 47%)) versus placebo. Laquinimod 1.0 mg showed less benefit (26.7% remission (CI 14% to 44%) and 53.3% response 70 (CI 36% to 70%)), and no effect was noted on remission/response at higher doses.
Laquinimod was safe and well tolerated, and the effects on remission and response of the 0.5 mg dose suggest a treatment benefit in patients with CD.
Recent data have suggested that non-selective β-blockers (NSBB) are associated with increased mortality in patients with cirrhosis and refractory ascites. However, other evidence implies that NSBB may be beneficial in this setting by reducing bacterial translocation. Our aim was to determine whether NSBB use was a risk factor for mortality in patients with end-stage chronic liver disease and ascites awaiting liver transplantation.
This was a single-centre retrospective study of 322 patients with ascites listed January 2007 to July 2011.
NSBB patients (n=159) and non-NSBB patients (n=163) were comparable with regards to listing model for end-stage liver disease score (p=0.168), frequency of hepatocellular carcinoma (p=0.193) and refractory ascites (35.2% vs. 37.4%, p=0.681). 82 patients died, 221 patients were transplanted and 19 patients were removed from the list during a median follow-up duration of 72 days; the median time to death was 150 and 54 days in the NSBB and non-NSBB groups, respectively. In a multivariate competing risk Cox model, patients on NSBB had reduced mortality compared with propensity risk score-matched non-NSBB patients (HR 0.55; 95% CI 0.32 to 0.95, p=0.032). Similarly, in the subgroup of patients with refractory ascites (n=117), NSBB remained independently associated with less waitlist death (adjusted HR 0.35; 95% CI 0.14 to 0.86, p=0.022).
NSBB in patients with ascites and refractory ascites listed for liver transplantation are not detrimental, and instead are associated with reduced waitlist death. Our findings argue that NSBB are safe and may confer benefit in patients with ascites complicating end-stage liver disease.
To assess variation in the main colonoscopy quality indicators in organised colorectal cancer (CRC) screening programmes based on faecal immunochemical test (FIT).
Data from a case-series of colonoscopies of FIT-positive subjects were provided by 44 Italian CRC screening programmes. Data on screening history, endoscopic procedure and histology results, and additional information on the endoscopy centre and the endoscopists were collected. The adenoma detection rate (ADR) and caecal intubation rate (CIR) were assessed for the whole population and the individual endoscopists. To explore variation in the quality indicators, multilevel analyses were performed according to patient/centre/endoscopist characteristics.
We analysed 75 569 (mean age: 61.3 years; men: 57%) colonoscopies for positive FIT performed by 479 endoscopists in 79 centres. ADR ranged from 13.5% to 75% among endoscopists (mean: 44.8%). ADR was associated with gastroenterology specialty (OR: 0.87 for others, 95% CI 0.76 to 0.96) and, at the endoscopy centre level, with the routine use of sedation (OR: 0.80 if occasional (<33%); 95% CI 0.64 to 1.00) and availability of screening-dedicated sessions (OR: 1.35; 95% CI 1.11 to 1.66). CIR ranged between 58.8% and 100% (mean: 93.1%). Independent predictors of CIR at the endoscopist level were the yearly number of screening colonoscopies performed (OR: 1.51 for endoscopists with >600 colonoscopies; 95% CI 1.11 to 2.04) and, at the endoscopy centre level, screening-dedicated sessions (OR: 2.18; 95% CI 1.24 to 3.83) and higher rates of sedation (OR: 0.47 if occasional; 95% CI 0.24 to 0.92).
The quality of colonoscopy was affected by patient-related, endoscopist-related and centre-related characteristics. Policies addressing organisational issues should improve the quality of colonoscopy in our programme and similar programmes.
Crohn's disease (CD) is caused by a complex interplay among genetic, microbial and environmental factors. ATG16L1 is an important genetic factor involved in innate immunity, including autophagy and phagocytosis of microbial components from the gut. We investigated the effect of inflammation on the composition of microbiota in the ileal mucosa of CD patients in relation to the ATG16L1 risk status.
Biopsies (n=35) were obtained from inflamed and non-inflamed regions of the terminal ileum of 11 CD patients homozygous for the ATG16L1 risk allele (ATG16L1-T300A) and 9 CD patients homozygous for the ATG16L1 protective allele (ATG16L1-T300). Biopsy DNA was extracted and the bacterial composition analysed by pyrosequencing. Intracellular survival rates of adherent-invasive Escherichia coli (AIEC) were analysed by determining colony forming units after exposure to monocytes isolated from healthy volunteers homozygous for the ATG16L1 risk or protective allele.
Inflamed ileal tissue from patients homozygous for the ATG16L1 risk allele contained increased numbers of Fusobacteriaceae, whereas inflamed ileal tissue of patients homozygous for the ATG16L1 protective allele showed decreased numbers of Bacteroidaceae and Enterobacteriaceae and increased Lachnospiraceae. The ATG16L1 allele did not affect the bacterial composition in the non-inflamed ileal tissue. Monocytes homozygous for the ATG16L1 risk allele showed impaired killing of AIEC under inflammatory conditions compared with those homozygous for the ATG16L1 protective allele.
CD patients homozygous for the ATG16L1–T300A risk allele show impaired clearance of pathosymbionts in ileal inflammation indicating that ATG16L1 is essential for effective elimination of pathosymbionts upon inflammation.
IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies.
We conducted a GWA study (GWAS) of IBS in a general population sample of 11 326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls.
One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31x10–6 in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls.
Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.
Assessment of disease activity in UC is important for designing an optimal therapeutic strategy. No single histology score is considered optimum. The aim of this study was to compare intraobserver reproducibility and the interobserver agreement of available histological UC activity indexes.
One hundred and two biopsy specimens (collected between 2003 and 2014) were scored blindly by three pathologists by determining Geboes, Riley, Gramlich and Gupta indexes and global visual evaluation (GVE). Intraobserver reproducibility and interobserver agreements for index and items of index were studied by intraclass correlation coefficient for quantitative parameter and by values and Krippendorff index for qualitative parameters. Relationship between indexes was studied by computation of Pearson's and Spearman's correlation coefficients.
Geboes, Riley, Gramlich and Gupta indexes and GVE showed good intraobserver reproducibility and a good interobserver agreement. Histological items that showed the best interobserver agreement were 'erosion/ulceration or surface epithelial integrity' and 'acute inflammatory cells infiltrate/neutrophils in lamina propria'. The five scores were strongly correlated.
Correlation between indexes is strong. Intraobserver reproducibility and interobserver agreement for all indexes is very good. Histological items that showed the best interobserver agreement are 'erosion/ulceration' and 'acute inflammatory cells infiltrate/neutrophils in lamina propria'.
The objective of this meta-analysis was to systematically assess the survival benefit of aspirin use before or after diagnosis for patients with colorectal cancer (CRC).
Relevant studies were identified through searching PubMed, Embase and Cochrane databases before May 2014. Two investigators extracted data independently for baseline characteristics and outcomes from the included studies. Either a fixed-effects or a random-effects model was derived to composite the pooled HR for overall mortality and CRC-specific mortality of CRC.
Seven studies on postdiagnosis aspirin therapy and seven studies on prediagnosis aspirin use were finally included in this meta-analysis. The overall survival benefit associated with postdiagnosis aspirin use represented an HR of 0.84 (95% CI 0.75 to 0.94). This effect was observed both in colon cancer (HR=0.78, 95% CI 0.64 to 0.96) and in rectal cancer (HR=0.90, 95% CI 0.83 to 0.98). Besides, the survival benefit of postdiagnosis aspirin use appeared to be confined to those patients with positive prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase-2, COX-2) expression (HR=0.65, 95% CI 0.50 to 0.85) and with mutated PIK3CA tumours (HR=0.58, 95% CI 0.37 to 0.90). Aspirin use postdiagnosis was not associated with CRC-specific mortality (HR=0.77, 95% CI 0.52 to 1.14). We observed no evidence of an association between prediagnosis aspirin use and CRC overall mortality (HR=1.01, 95% CI 0.96 to 1.06) or CRC-specific mortality (HR=0.93, 95% CI 0.82 to 1.05).
These findings provide further indication that postdiagnosis aspirin therapy improved CRC overall survival, especially for patients with positive PTGS2 (COX-2) expression and mutated PIK3CA tumours.
We aimed to determine the predictors and risk for death among persons with either Crohn's disease (CD) or UC compared with the general population.
We used the population-based University of Manitoba IBD Epidemiology Database to calculate HRs and their 95% CIs for cases relative to controls using stratified multivariable Cox proportional hazards regression models, controlling for socioeconomic status and comorbidities.
There were 10 788 prevalent cases of CD and UC and 101 860 matched controls. The HR for all-cause mortality in prevalent CD cases was 1.26 (95% CI 1.16 to 1.38) and in prevalent UC cases was 1.04 (95% CI 0.96 to 1.12). Compared with matched controls, CD cases were more likely to die of colorectal cancer, non-Hodgkin's lymphoma, digestive diseases, pulmonary embolism and sepsis and UC cases were more likely to die from colorectal cancer, digestive diseases and respiratory diseases. For incident cases, there were significant effects on mortality by socioeconomic status, comorbidity score and surgery. The greatest risk for death in both CD and UC was within the first 30 days following GI surgery. The first year from diagnosis was associated with increased risk of death in both CD and UC, but persisted after the 1st year only in CD.
There is a significantly increased risk of mortality in CD compared with controls while in UC an increased risk for death was only evident in the first year from diagnosis. Surgery poses an increased risk for death in both CD and UC lasting up to 1 year.
The rising incidence of inflammatory bowel disease in Asia supports the importance of environmental risk factors in disease aetiology. This prospective population-based case-control study in Asia-Pacific examined risk factors prior to patients developing IBD.
442 incident cases (186 Crohn's disease (CD); 256 UC; 374 Asians) diagnosed between 2011 and 2013 from eight countries in Asia and Australia and 940 controls (frequency-matched by sex, age and geographical location; 789 Asians) completed an environmental factor questionnaire at diagnosis. Unconditional logistic regression models were used to estimate adjusted ORs (aOR) and 95% CIs.
In multivariate model, being breast fed >12 months (aOR 0.10; 95% CI 0.04 to 0.30), antibiotic use (aOR 0.19; 0.07 to 0.52), having dogs (aOR 0.54; 0.35 to 0.83), daily tea consumption (aOR 0.62; 0.43 to 0.91) and daily physical activity (aOR 0.58; 0.35 to 0.96) decreased the odds for CD in Asians. In UC, being breast fed >12 months (aOR 0.16; 0.08 to 0.31), antibiotic use (aOR 0.48; 0.27 to 0.87), daily tea (aOR 0.63; 0.46 to 0.86) or coffee consumption (aOR 0.51; 0.36 to 0.72), presence of hot water tap (aOR 0.65; 0.46 to 0.91) and flush toilet in childhood (aOR 0.71; 0.51 to 0.98) were protective for UC development whereas ex-smoking (aOR 2.02; 1.22 to 3.35) increased the risk of UC.
This first population-based study of IBD risk factors in Asia-Pacific supports the importance of childhood immunological, hygiene and dietary factors in the development of IBD, suggesting that markers of altered intestinal microbiota may modulate risk of IBD later in life.
The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C.
We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥20 g/day for ≥5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR.
Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR.
Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.
The aim was to determine the prevalence of small-bowel neoplasia in asymptomatic patients with Lynch syndrome (LS) by video capsule endoscopy (VCE).
After obtaining informed consent, asymptomatic proven gene mutation carriers aged 35–70 years were included in this prospective multicentre study in the Netherlands. Patients with previous small-bowel surgery were excluded. After bowel preparation, VCE was performed. The videos were read by two independent investigators. If significant lesions were detected, an endoscopic procedure was subsequently performed to obtain histology and, if possible, remove the lesion.
In total, 200 patients (mean age 50 years (range 35–69), M/F 88/112), with proven mutations were included. These concerned MLH1 (n=50), MSH2 (n=68), MSH6 (n=76), PMS2 (n=3) and Epcam (n=3) mutation carriers. In 95% of the procedures, caecal visualisation was achieved. Small-bowel neoplasia was detected in two patients: one adenocarcinoma (TisN0Mx) and one adenoma, both located in the duodenum. In another patient, a duodenal cancer (T2N0Mx) was diagnosed 7 months after a negative VCE. This was considered a lesion missed by VCE. All three neoplastic lesions were within reach of a conventional gastroduodenoscope. All patients with neoplasia were men, over 50 years of age and without a family history of small-bowel cancer.
The prevalence of small-bowel neoplasia in asymptomatic patients with LS was 1.5%. All neoplastic lesions were located in the duodenum and within reach of conventional gastroduodenoscopy. Although VCE has the potential to detect these neoplastic lesions, small-bowel neoplasia may be missed.
Eosinophils reside in the colonic mucosa and increase significantly during disease. Although a number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the expanding scope of eosinophil-mediated activities indicate that they also regulate local immune responses and modulate tissue inflammation. We sought to define the impact of eosinophils that respond to acute phases of colitis in mice.
Acute colitis was induced in mice by administration of dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid or oxazolone to C57BL/6J (control) or eosinophil deficient (PHIL) mice. Eosinophils were also depleted from mice using antibodies against interleukin (IL)-5 or by grafting bone marrow from PHIL mice into control mice. Colon tissues were collected and analysed by immunohistochemistry, flow cytometry and reverse transcription PCR; lipids were analysed by mass spectroscopy.
Eosinophil-deficient mice developed significantly more severe colitis, and their colon tissues contained a greater number of neutrophils, than controls. This compensatory increase in neutrophils was accompanied by increased levels of the chemokines CXCL1 and CXCL2, which attract neutrophils. Lipidomic analyses of colonic tissue from eosinophil-deficient mice identified a deficiency in the docosahexaenoic acid-derived anti-inflammatory mediator 10, 17- dihydroxydocosahexaenoic acid (diHDoHE), namely protectin D1 (PD1). Administration of an exogenous PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of colitis in eosinophil-deficient mice. The PD1-isomer also attenuated neutrophil infiltration and reduced levels of tumour necrosis factor-α, IL-1β, IL-6 and inducible NO-synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration.
Eosinophils exert a protective effect in acute mouse colitis, via production of anti-inflammatory lipid mediators.
Altered intestinal barrier is associated with immune activation and clinical symptoms in diarrhoea-predominant IBS (IBS-D). Increased mucosal antigen load may induce specific responses; however, local antibody production and its contribution to IBS aetiopathogenesis remain undefined. This study evaluated the role of humoral activity in IBS-D.
A single mucosal jejunal biopsy, luminal content and blood were obtained from healthy volunteers (H; n=30) and IBS-D (n=49; Rome III criteria) participants. Intraepithelial lymphocytes, mast cells, B lymphocytes and plasma cells were studied by imaging techniques. Differential gene expression and pathway analysis were assessed by microarray and PCR techniques. Blood and luminal immunoglobulins (Igs) were quantified. Gastrointestinal symptoms, respiratory atopy and stress and depression were also recorded.
Patients with IBS-D showed a higher number and activation of mucosal B lymphocytes and plasma cells (p<0.05). Mast cell density was increased in patients with IBS-D (non-atopic) and in close proximity to plasma cells (p<0.05). Microarray profiling identified differential humoral activity in IBS-D, involving proliferation and activation of B lymphocytes and Igs production (p<0.001). Mucosal humoral activity was higher in IBS-D, with upregulation of germline transcripts and Ig genes (1.3-fold–1.7-fold increase; p<0.05), and increased IgG+ cells and luminal IgG compared with H (p<0.05), with no differences in blood. Biological markers of humoral activity correlated positively with bowel movements, stool form and depression.
Enhanced small bowel humoral immunity is a distinctive feature of IBS-D. Mucosal Ig production contributes to local inflammation and clinical manifestations in IBS-D.
The identification of ATG16L1 as a CD susceptibility gene has suggested...]]>
We read with interest the paper by Amaddeo et al
In parallel, in a very recent Cancer Cell paper, Lau et al
Interval colorectal cancers (interval CRCs), that is, cancers occurring after a negative screening test or examination, are an important indicator of the quality and effectiveness of CRC screening and surveillance. In order to compare incidence rates of interval CRCs across screening programmes, a standardised definition is required. Our goal was to develop an internationally applicable definition and taxonomy for reporting on interval CRCs.
Using a modified Delphi process to achieve consensus, the Expert Working Group on interval CRC of the Colorectal Cancer Screening Committee of the World Endoscopy Organization developed a nomenclature for defining and characterising interval CRCs.
We define an interval CRC as a "colorectal cancer diagnosed after a screening or surveillance exam in which no cancer is detected, and before the date of the next recommended exam". Guidelines and principles for describing and reporting on interval CRCs are provided, and clinical scenarios to demonstrate the practical application of the nomenclature are presented.
The Working Group on interval CRC of the World Endoscopy Organization endorses adoption of this standardised nomenclature. A standardised nomenclature will facilitate benchmarking and comparison of interval CRC rates across programmes and regions.
We read with great interest the leading article by Bruix et al
With better patient selection and improvement of perioperative care, liver resection (LR) offers the most consistent and clinically meaningful long-term survival in HCC over the past 20 years, which has been documented by both Eastern and Western centres.
The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in hepatocellular carcinoma (HCC), but its biological role has not been clarified. In this study, we explored the role of ERK5 expression and activity in HCC in vitro and in vivo.
ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacological inhibitor, XMD8-92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts.
In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue compared with normal liver tissue. Inhibition of ERK5 decreased HCC cell proliferation and increased the proportion of cells in G0/G1 phase. These effects were associated with increased expression of p27 and p15 and decreased CCND1. Treatment with XMD8-92 or ERK5 silencing prevented cell migration induced by epidermal growth factor or hypoxia and caused cytoskeletal remodelling. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 was silenced for ERK5. Moreover, systemic treatment with XMD8-92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel.
ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC.
In this issue of Gut, VanDussen et al
The authors established a very simple methodology...]]>
There is uncertainty whether cancer screening affects participant incentives for favourable lifestyle. The present study investigates long-term effects of colorectal cancer (CRC) screening on lifestyle changes.
In 1999–2001, men and women drawn from the population registry were randomised to screening for CRC by flexible sigmoidoscopy ('invited-to-screening' arm) or to no-screening (control arm) in the Norwegian Colorectal Cancer Prevention trial. A subgroup of 3043 individuals in the 'invited-to-screening' and 2819 in the control arm, aged 50–55 years, randomised during 2001 had their lifestyle assessed by a questionnaire at inclusion and after 11 years (42% of cohort). The outcome was 11-year changes in lifestyle factors (body weight, smoking status, physical exercise, selected dietary habits) and in total lifestyle score (0–4 points, translating to the number of lifestyle recommendations adhered to). We compared outcomes in the two randomisation arms and attendees with positive versus negative findings.
Total lifestyle scores improved in both arms. The improvement was smaller in the 'invited-to-screening' arm (score 1.43 at inclusion; 1.58 after 11 years) compared with the control arm (score 1.49 at inclusion; 1.67 after 11 years); adjusted difference –0.05 (95% CI –0.09 to –0.01; p=0.03). The change in the score was less favourable in screening attendees with a positive compared with negative screening result; adjusted difference –0.16 (95% CI –0.25 to –0.08; p<0.001).
The present study suggests that possible unfavourable lifestyle changes after CRC screening are modest. Lifestyle counselling may be considered as part of cancer screening programmes.
Liver disease is an increasing clinical burden, causing over 10 000 deaths last year in the UK alone (http://www.britishlivertrust.org.uk). Liver insufficiency describes the clinical situation in which cumulative (chronic) or one-off massive (acute) insults exceed the liver's normal physiological capacity to functionally regenerate. Untreated, liver insufficiency invariably leads to death. The current gold standard of care in this setting is whole organ transplantation. Due to the increasing burden of disease within the population, however, the number of patients requiring transplantation far exceeds the number of available donor organs. As a result, many patients with liver insufficiency die prematurely.
The liver is composed of several cell types (endothelial cells, stellate cells, biliary ductal cells, Kupffer cells and natural killer cells) which together provide a supportive niche for the principle cell type—the hepatocyte.
Interleukin-26 (IL-26) is a member of the IL-10 cytokine family, first discovered based on its peculiar expression by virus-transformed T cells. IL-26 is overexpressed in chronic inflammation (rheumatoid arthritis and Crohn's disease) and induces proinflammatory cytokines by myeloid cells and some epithelial cells. We thus investigated the expression and potential role of IL-26 in chronic HCV infection, a pathology associated with chronic inflammation.
IL-26 was quantified in a cohort of chronically HCV-infected patients, naive of treatment and its expression in the liver biopsies investigated by immunohistochemistry. We also analysed the ability of IL-26 to modulate the activity of natural killer (NK) cells, which control HCV infection.
The serum levels of IL-26 are enhanced in chronically HCV-infected patients, mainly in those with severe liver inflammation. Immunohistochemistry reveals an intense IL-26 staining in liver lesions, mainly in infiltrating CD3+ cells. We also show that NK cells from healthy subjects and from HCV-infected patients are sensitive to IL-26. IL-26 upregulates membrane tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression on CD16– CD56bright NK cells, enabling them to kill HCV-infected hepatoma cells, with the same efficacy as interferon (IFN)-α-treated NK cells. IL-26 also induces the expression of the antiviral cytokines IFN-β and IFN-, and of the proinflammatory cytokines IL-1β and TNF-α by NK cells.
This study highlights IL-26 as a new player in the inflammatory and antiviral immune responses associated with chronic HCV infection.
To compare the performance of existing sigmoidoscopy-based strategies in predicting advanced proximal neoplasia (APN) in an asymptomatic Chinese cohort.
We included all screening participants aged 50–70 years who received colonoscopy between 2008 and 2014 in Hong Kong. Sigmoidoscopy yield was estimated from the colonoscopic findings based on the: (1) UK flexible sigmoidoscopy; (2) Screening for COlon REctum (SCORE); (3) NORwegian Colorectal Cancer Prevention (NORCCAP) trials and (4) US clinical index based on age, gender and distal findings. The sensitivity, specificity, the number of subjects needed to screen (NNS) and the number of subjects needed to refer (NNR) for colonoscopy to detect one APN were evaluated. Binary logistic regression modelling identified the distal findings associated with APN.
From 5879 eligible subjects, 132 (2.2%) had APN. The US strategy achieved the highest sensitivity for APN detection (42.0%) and the UK criteria attained the highest specificity (96%). The US criteria led to the lowest NNS (92 vs 103–267) and the UK criteria required the least NNR (12 vs 16–21). Using the US strategy, the rates of APN detected were 1.4% (low-risk group), 2.2% (intermediate risk) and 5.9% (high risk). The c-statistics of the UK, SCORE, NORCCAP and the US criteria were 0.55±0.03; 0.59±0.03; 0.59±0.03 and 0.62±0.05 respectively.
The US criteria had the highest sensitivity for detection of APN and lowest NNS and the UK score had the highest specificity and the lowest NNR. The performance of all these four criteria to predict APN is limited, highlighting an urgent need to devise a novel APN prediction system for Asian subjects.
Colorectal cancer (CRC) screening programmes based on the guaiac faecal occult blood test (gFOBT) reduce CRC-specific mortality. Several studies have shown higher sensitivity with the faecal immunochemical test (FIT) compared with gFOBT. We carried out an ecological study to evaluate the impact of FIT-based screening programmes on CRC mortality.
In the Veneto Region (Italy), biennial FIT-based screening programmes that invited 50–69-year-old residents were introduced in different areas between 2002 and 2009. We compared CRC mortality rates from 1995 to 2011 between the areas where screening started in 2002–2004 (early screening areas (ESA)) and areas that introduced the screening in 2008–2009 (late screening areas (LSA)) using Poisson regression models. We also compared available data on CRC incidence rates (1995–2007) and surgical resection rates (2001–2012).
Before the introduction of screening, CRC mortality and incidence rates in the two areas were similar. Compared with 1995–2000, 2006–2011 mortality rates were 22% lower in the ESA than in the LSA (rate ratio (RR)=0.78; 95% CI 0.68 to 0.89). The reduction was larger in women (RR=0.64; CI 0.51 to 0.80) than in men (RR=0.87; CI 0.73 to 1.04). In the ESA, incidence and surgery rates peaked during the introduction of the screening programme and then returned to the baseline (2006–2007 incidence) or dropped below initial values (surgery after 2007).
FIT-based screening programmes were associated with a significant reduction in CRC mortality. This effect took place much earlier than reported by gFOBT-based trials and observational studies.
Helicobacter pylori colonisation rates in childhood have declined in Western populations, but it is unknown whether this trend is similar in children of non-Western ethnic backgrounds, born in a Western country. We aimed to identify H. pylori status in children, and determine mother-to-child transmission and risk factors for colonisation.
Antibodies against H. pylori and cytotoxin-associated gene A (CagA) were measured in children participating in a population-based prospective cohort study in Rotterdam, the Netherlands. Information on demographics and characteristics was collected using questionnaires.
We analysed the serum of 4467 children (mean age 6.2 years±0.4 SD) and compared the results with the H. pylori status of their mothers (available for 3185 children). Overall, 438 (10%) children were H. pylori-positive, of whom 142 (32%) were CagA-positive. Independent risk factors for colonisation were: maternal H. pylori positivity (OR 2.12; 95% CI 1.62 to 2.77), non-Dutch ethnicity (OR 2.05; 95% CI 1.54 to 2.73), female gender (OR 1.47; 95% CI 1.20 to 1.80) and lower maternal education level (OR 1.38; 95% CI 1.06 to 1.79). Comparing mothers and children, we found an intergenerational decrease of 76% and 77% for Hp+CagA– and Hp+CagA+-strains, respectively, consistent across all nine ethnic groups studied. Male gender, higher maternal educational level and no older siblings, were independently associated with absence of H. pylori.
Although the highest H. pylori and CagA prevalence was found in children of non-Dutch ethnicities, the decreased colonisation rates were uniform across all ethnic groups, implying the importance of environmental factors in H. pylori transmission in modern cities, independent of ethnicity.
Compared with a significant post-treatment stricture rate (>30% for PDT
Many cross-sectional surveys have reported the prevalence of uninvestigated dyspepsia, but there has been no recent systematic review of data from all studies to determine its global prevalence and risk factors.
MEDLINE, EMBASE and EMBASE Classic were searched (until January 2014) to identify population-based studies that reported the prevalence of uninvestigated dyspepsia in adults (≥15 years old); dyspepsia was defined using symptom-based criteria or questionnaires. The prevalence of dyspepsia was extracted for all studies and according to the criteria used to define it. Pooled prevalence, according to study location and certain other characteristics, ORs and 95% CIs were calculated.
Of the 306 citations evaluated, 103 reported the prevalence of uninvestigated dyspepsia in 100 separate study populations, containing 312 415 subjects. Overall pooled prevalence in all studies was 20.8% (95% CI 17.8% to 23.9%). The prevalence varied according to country (from 1.8% to 57.0%) and criteria used to define dyspepsia. The greatest prevalence values were found when a broad definition of dyspepsia (29.5%; 95% CI 25.3% to 33.8%) or upper abdominal or epigastric pain or discomfort (20.4%; 95% CI 16.3% to 24.8%) were used. The prevalence was higher in women (OR 1.24; 95% CI 1.13 to 1.36), smokers (OR 1.25; 95% CI 1.12 to 1.40), non-steroidal anti-inflammatory drug (NSAID) users (OR 1.59; 95% CI 1.27 to 1.99) and Helicobacter pylori-positive individuals (OR 1.18; 95% CI 1.04 to 1.33).
The overall pooled prevalence of uninvestigated dyspepsia was 21%, but varied among countries and according to the criteria used to define its presence. Prevalence is significantly higher in women, smokers, NSAID users and H. pylori-positive individuals, although these associations were modest.
The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression.
We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100 000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years).
In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child–Turcotte–Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001).
A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.
In-home iron fortification for infants in developing countries is recommended for control of anaemia, but low absorption typically results in >80% of the iron passing into the colon. Iron is essential for growth and virulence of many pathogenic enterobacteria. We determined the effect of high and low dose in-home iron fortification on the infant gut microbiome and intestinal inflammation.
We performed two double-blind randomised controlled trials in 6-month-old Kenyan infants (n=115) consuming home-fortified maize porridge daily for 4 months. In the first, infants received a micronutrient powder (MNP) containing 2.5 mg iron as NaFeEDTA or the MNP without iron. In the second, they received a different MNP containing 12.5 mg iron as ferrous fumarate or the MNP without the iron. The primary outcome was gut microbiome composition analysed by 16S pyrosequencing and targeted real-time PCR (qPCR). Secondary outcomes included faecal calprotectin (marker of intestinal inflammation) and incidence of diarrhoea. We analysed the trials separately and combined.
At baseline, 63% of the total microbial 16S rRNA could be assigned to Bifidobacteriaceae but there were high prevalences of pathogens, including Salmonella Clostridium difficile, Clostridium perfringens, and pathogenic Escherichia coli. Using pyrosequencing, +FeMNPs increased enterobacteria, particularly Escherichia/Shigella (p=0.048), the enterobacteria/bifidobacteria ratio (p=0.020), and Clostridium (p=0.030). Most of these effects were confirmed using qPCR; for example, +FeMNPs increased pathogenic E. coli strains (p=0.029). +FeMNPs also increased faecal calprotectin (p=0.002). During the trial, 27.3% of infants in +12.5 mgFeMNP required treatment for diarrhoea versus 8.3% in –12.5 mgFeMNP (p=0.092). There were no study-related serious adverse events in either group.
In this setting, provision of iron-containing MNPs to weaning infants adversely affects the gut microbiome, increasing pathogen abundance and causing intestinal inflammation.
Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known crosstalk between VD and transforming growth factor (TGF)-β signalling, VD has been proposed as an antifibrotic treatment.
We evaluated the association between VD, the vitamin D receptor (VDR) and liver fibrosis in primary human hepatic stellate cells (phHSC) and 106 morbidly obese patients with NAFLD.
Treating phHSC with VD ameliorated TGF-β-induced fibrogenesis via both VDR-dependent and VDR-independent mechanisms. Reduction of fibrogenic response was abolished in cells homozygous for GG at the A1012G single nucleotide polymorphisms within the VDR gene. Compared with healthy livers, NAFLD livers expressed higher levels of VDR mRNA and VDR fragments. VDR mRNA was lower in patients homozygous for GG at A1012G and expression of pro-fibrogenic genes was higher in patients carrying the G allele.
VD may be an antifibrotic treatment option early in the onset of fibrosis in specific genotypes for VDR. Known polymorphisms of the VDR may influence the response to VD treatment.
Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown.
Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays.
Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis.
This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.
Helicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag+ strains heighten cancer risk have not been fully delineated and model systems that recapitulate the gastric niche are critical for understanding pathogenesis. Gastroids are three-dimensional organ-like structures that provide unique opportunities to study host-H. pylori interactions in a preclinical model. We used gastroids to inform and direct in vitro studies to define mechanisms through which H. pylori modulates expression of the cancer-associated tight junction protein claudin-7.
Gastroids were infected by luminal microinjection, and MKN28 gastric epithelial cells were cocultured with H. pylori wild-type cag+ strains or isogenic mutants. β-catenin, claudin-7 and snail localisation was determined by immunocytochemistry. Proliferation was assessed using 5-ethynyl-2'-deoxyuridine, and levels of claudin-7 and snail were determined by western blot and flow cytometry.
Gastroids developed into a self-organising differentiation axis and H. pylori induced mislocalisation of claudin-7 and increased proliferation in a CagA- and β-catenin-dependent manner. In MKN28 cells, H pylori-induced suppression of claudin-7 was regulated by β-catenin and snail. Similarly, snail expression was increased and claudin-7 levels were decreased among H. pylori-infected individuals.
H. pylori increase proliferation in a strain-specific manner in a novel gastroid system. H. pylori also alter expression and localisation of claudin-7 in gastroids and human epithelial cells, which is mediated by β-catenin and snail activation. These data provide new insights into molecular interactions with carcinogenic potential that occur between H. pylori and epithelial cells within the gastric niche.
Neurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo.
miR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro.
NT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-B activation, as well as IL-6, IL-8 and IL-1β messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls.
NT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.
In this study, we investigated the role of Trefoil factor 1 (TFF1) in regulating cell proliferation and tumour development through β-catenin signalling using in vivo and in vitro models of gastric tumorigenesis.
Tff1-knockout (Tff1-KO) mice, immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot, and phosphatase assays were used to examine the role of TFF1 on β-catenin signalling pathway.
Nuclear localisation of β-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at an early age of 4–6 weeks and maintained during all stages of gastric tumorigenesis in the Tff1-KO mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the β-catenin/T-cell factor (TCF) transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear β-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signalling molecules upstream of β-catenin revealed a decrease in phosphorylated glycogen synthase kinase 3β (p-GSK3β) (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-β-catenin (Ser33/37/Thr41) and decrease of p-β-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3β, and AKT was dependent on protein phosphatase 2A (PP2A) activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localisation of β-catenin in stages of human gastric tumorigenesis.
Our data indicate that loss of TFF1 promotes β-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3β signalling.
In 2014, three drugs were approved in the European Union, including the nucleotide analogue sofosbuvir, the protease inhibitor simeprevir and the NS5A inhibitor daclatasvir. They can be used in...]]>
Caspase-2 is an initiator caspase involved in multiple apoptotic pathways, particularly in response to specific intracellular stressors (eg, DNA damage, ER stress). We recently reported that caspase-2 was pivotal for the induction of cell death triggered by excessive intracellular accumulation of long-chain fatty acids, a response known as lipoapoptosis. The liver is particularly susceptible to lipid-induced damage, explaining the pandemic status of non-alcoholic fatty liver disease (NAFLD). Progression from NAFLD to non-alcoholic steatohepatitis (NASH) results, in part, from hepatocyte apoptosis and consequential paracrine-mediated fibrogenesis. We evaluated the hypothesis that caspase-2 promotes NASH-related cirrhosis.
Caspase-2 was localised in liver biopsies from patients with NASH. Its expression was evaluated in different mouse models of NASH, and outcomes of diet-induced NASH were compared in wild-type (WT) and caspase-2-deficient mice. Lipotoxicity was modelled in vitro using hepatocytes derived from WT and caspase-2-deficient mice.
We showed that caspase-2 is integral to the pathogenesis of NASH-related cirrhosis. Caspase-2 is localised in injured hepatocytes and its expression was markedly upregulated in patients and animal models of NASH. During lipotoxic stress, caspase-2 deficiency reduced apoptosis, inhibited induction of profibrogenic hedgehog target genes in mice and blocked production of hedgehog ligands in cultured hepatocytes.
These data point to a critical role for caspase-2 in lipid-induced hepatocyte apoptosis in vivo for the production of apoptosis-associated fibrogenic factors and in the progression of lipid-induced liver fibrosis. This raises the intriguing possibility that caspase-2 may be a promising therapeutic target to prevent progression to NASH.
To study the detailed nature of genomic microevolution during mixed infection with multiple Helicobacter pylori strains in an individual.
We sampled 18 isolates from a single biopsy from a patient with chronic gastritis and nephritis. Whole-genome sequencing was applied to these isolates, and statistical genetic tools were used to investigate their evolutionary history.
The genomes fall into two clades, reflecting colonisation of the stomach by two distinct strains, and these lineages have accumulated diversity during an estimated 2.8 and 4.2 years of evolution. We detected about 150 clear recombination events between the two clades. Recombination between the lineages is a continuous ongoing process and was detected on both clades, but the effect of recombination in one clade was nearly an order of magnitude higher than in the other. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and we estimate that they have both been infecting the same host for at least 12 years. Recombination tracts between the lineages were, on average, 895 bp in length, and showed evidence for the interspersion of recipient sequences that has been observed in in vitro experiments. The complex evolutionary history of a phage-related protein provided evidence for frequent reinfection of both clades by a single phage lineage during the past 4 years.
Whole genome sequencing can be used to make detailed conclusions about the mechanisms of genetic change of H. pylori based on sampling bacteria from a single gastric biopsy.
To evaluate the safety and efficacy of daclatasvir, an HCV NS5A inhibitor with pangenotypic activity, administered with peginterferon-alfa-2a/ribavirin.
In this Phase 2b double-blind, placebo-controlled study, treatment-naive adults with HCV genotype 1 (N=365) or 4 (N=30) infection were randomly assigned (2:2:1) to daclatasvir 20 mg or 60 mg, or placebo once daily plus weekly peginterferon-alfa-2a and twice-daily ribavirin. Daclatasvir recipients achieving protocol-defined response (PDR; HCV-RNA<lower limit of quantitation at Week 4 and undetectable at Week 10) were rerandomised at Week 12 to continue daclatasvir/peginterferon-alfa-2a/ribavirin for 24 weeks total duration or to placebo/peginterferon-alfa-2a/ribavirin for another 12 weeks. Patients without PDR and placebo patients continued peginterferon-alfa/ribavirin through Week 48. Primary efficacy endpoints were undetectable HCV-RNA at Weeks 4 and 12 (extended rapid virologic response, eRVR) and at 24 weeks post-treatment (sustained virologic response, SVR24) among genotype 1-infected patients.
Overall, eRVR was achieved by 54.4% (80/147) of genotype 1-infected patients receiving daclatasvir 20 mg, 54.1% (79/146) receiving 60 mg versus 13.9% (10/72) receiving placebo. SVR24 was achieved among 87 (59.2%), 87 (59.6%), and 27 (37.5%) patients in these groups, respectively. Higher proportions of genotype 4-infected patients receiving daclatasvir 20 mg (66.7%; 8/12) or 60 mg (100.0%; 12/12) achieved SVR24 versus placebo (50.0%; 3/6). A majority of daclatasvir-treated patients achieved PDR and experienced less virologic failure and higher SVR24 rates with a shortened 24-week treatment duration. Adverse events occurred with similar frequency across all treatment groups.
The combination of daclatasvir/peginterferon-alfa/ribavirin was generally well tolerated and achieved higher SVR24 rates compared with placebo/peginterferon-alfa/ribavirin among patients infected with HCV genotype 1 or 4.
Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms of genes such as ATG16L1 have been associated with increased risk. Although vitamin D, the microbiome and autophagy are all involved in pathogenesis of IBD, it remains unclear whether these processes are related or function independently.
We investigated the effects and mechanisms of intestinal epithelial VDR in healthy and inflamed states using cell culture models, a conditional VDR knockout mouse model (VDRIEC), colitis models and human samples.
Absence of intestinal epithelial VDR affects microbial assemblage and increases susceptibility to dextran sulfate sodium-induced colitis. Intestinal epithelial VDR downregulates expressions of ATG16L1 and lysozyme, and impairs antimicrobial function of Paneth cells. Gain and loss-of-function assays showed that VDR levels regulate ATG16L1 and lysozyme at the transcriptional and translational levels. Moreover, low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and representation by intestinal Bacteroides in patients with IBD. Administration of the butyrate (a fermentation product of gut microbes) increases intestinal VDR expression and suppresses inflammation in a colitis model.
Our study demonstrates fundamental relationship between VDR, autophagy and gut microbial assemblage that is essential for maintaining intestinal homeostasis, but also in contributing to the pathophysiology of IBD. These insights can be leveraged to define therapeutic targets for restoring VDR expression and function.
Infection with Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the stomach. Tumorigenic transformation of gastric epithelium induced by H. pylori is a highly complex process driven by an active interplay between bacterial virulence and host factors, many aspects of which remain obscure. In this work, we investigated the degradation of p53 tumour suppressor induced by H. pylori.
Expression of p53 protein in gastric biopsies was assessed by immunohistochemistry. Gastric cells were co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas and assessed for expression of p53, p14ARF and cytotoxin-associated gene A (CagA) by immunoblotting. siRNA was used to inhibit activities of ARF-BP1 and Human Double Minute 2 (HDM2) proteins.
Our analysis demonstrated that H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p53 compared with low-risk strains in vivo and in vitro. We found that degradation of p53 induced by bacterial CagA protein is mediated by host HDM2 and ARF-BP1 E3 ubiquitin ligases, while the p14ARF protein counteracts H. pylori-induced signalling.
Our results provide novel evidence that tumorigenicity associated with H. pylori infection is linked to inhibition of p53 protein by CagA. We propose a model in which CagA-induced degradation of p53 protein is determined by a relative level of p14ARF. In cells in which p14ARF levels were decreased due to hypermethylation or deletion of the p14ARF gene, H. pylori efficiently degraded p53, whereas p53 is protected in cells expressing high levels of p14ARF.
The increasing prevalence of obesity and type 2 diabetes (T2D) demonstrates the failure of conventional treatments to curb these diseases. The gut microbiota has been put forward as a key player in the pathophysiology of diet-induced T2D. Importantly, cranberry (Vaccinium macrocarpon Aiton) is associated with a number of beneficial health effects. We aimed to investigate the metabolic impact of a cranberry extract (CE) on high fat/high sucrose (HFHS)-fed mice and to determine whether its consequent antidiabetic effects are related to modulations in the gut microbiota.
C57BL/6J mice were fed either a chow or a HFHS diet. HFHS-fed mice were gavaged daily either with vehicle (water) or CE (200 mg/kg) for 8 weeks. The composition of the gut microbiota was assessed by analysing 16S rRNA gene sequences with 454 pyrosequencing.
CE treatment was found to reduce HFHS-induced weight gain and visceral obesity. CE treatment also decreased liver weight and triglyceride accumulation in association with blunted hepatic oxidative stress and inflammation. CE administration improved insulin sensitivity, as revealed by improved insulin tolerance, lower homeostasis model assessment of insulin resistance and decreased glucose-induced hyperinsulinaemia during an oral glucose tolerance test. CE treatment was found to lower intestinal triglyceride content and to alleviate intestinal inflammation and oxidative stress. Interestingly, CE treatment markedly increased the proportion of the mucin-degrading bacterium Akkermansia in our metagenomic samples.
CE exerts beneficial metabolic effects through improving HFHS diet-induced features of the metabolic syndrome, which is associated with a proportional increase in Akkermansia spp. population.
With interest we read the paper by Juste et al
Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated.
To assess whether human HSCs are susceptible to HCV infection.
We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs.
In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication.
Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process.
In case of incomplete colonoscopy, several radiologic methods have traditionally been used, but more recently, capsule endoscopy was also shown to be accurate. Aim of this study was to compare colon capsule endoscopy (CCE) and CT colonography (CTC) in a prospective cohort of patients with incomplete colonoscopy.
Consecutive patients with a previous incomplete colonoscopy underwent CCE and CTC followed by colonoscopy in case of positive findings on either test (polyps/mass lesions ≥6 mm). Clinical follow-up was performed in the other cases to rule out missed cancer. CTC was performed after colon capsule excretion or 10–12 h postingestion. Since the gold standard colonoscopy was performed only in positive cases, diagnostic yield and positive predictive values of CCE and CTC were used as study end-points.
100 patients were enrolled. CCE and CTC were able to achieve complete colonic evaluation in 98% of cases. In a per-patient analysis for polyps ≥6 mm, CCE detected 24 patients (24.5%) and CTC 12 patients (12.2%). The relative sensitivity of CCE compared to CTC was 2.0 (95% CI 1.34 to 2.98), indicating a significant increase in sensitivity for lesions ≥6 mm. Of larger polyps (≥10 mm), these values were 5.1% for CCE and 3.1% for CTC (relative sensitivity: 1.67 (95% CI 0.69 to 4.00)). Positive predictive values for polyps ≥6 mm and ≥10 mm were 96% and 85.7%, and 83.3% and 100% for CCE and CTC, respectively. No missed cancer occurred at clinical follow-up of a mean of 20 months.
CCE and CTC were of comparable efficacy in completing colon evaluation after incomplete colonoscopy; the overall diagnostic yield of colon capsule was superior to CTC.
Pepsin may be detected in sputum or saliva by enzymatic or immunological tests.
Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium.
Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems.
Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-B pathway and upregulation of the novel NF-B inhibitor SLPI but no other known NF-B inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-B activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs.
We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.
Immune tolerance breakdown during UC involves the peroxisome proliferator-activated receptor- (PPAR), a key factor in mucosal homoeostasis and the therapeutic target of 5-aminosalycilates, which expression is impaired during UC. Here we assess the impact of glucocorticoids (GCs) on PPAR expression, focusing especially on extra-adrenal cortisol production by colonic epithelial cells (CECs).
Activation of PPAR in the colon was evaluated using transgenic mice for the luciferase gene under PPAR control (peroxisome proliferator response element-luciferase mice). Protein and mRNA expression of PPAR were evaluated with colon fragments and purified CEC from mice. Cortisol production and steroidogenic factor expression were quantified in human CEC of patients with UC and those of controls. Gene expression knockdown by short hairpin RNA in Caco-2 cells was used for functional studies.
GCs were able to raise luciferase activity in peroxisome proliferator response element-luciferase mice. In the mice colons and Caco-2 cells, PPAR expression was increased either with GCs or with an inducer of steroidogenesis and then decreased after treatment with a steroidogenesis inhibitor. Cortisol production and steroidogenic factor expression, such as liver receptor homologue-1 (LRH-1), were decreased in CEC isolated from patients with UC, directly correlating with PPAR impairment. Experiments on Caco-2 cells lacking LRH-1 expression confirmed that LRH-1 controls PPAR expression by regulating GC synthesis in CEC.
These results demonstrate cortisol control of PPAR expression in CEC, highlighting cortisol production deficiency in colonocytes as a key molecular event in the pathophysiology of UC.
Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications.
KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice.
KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs.
KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
Surveillance is recommended for Barrett's oesophagus (BO) to detect early oesophageal adenocarcinoma (OAC). The aim of this study was to evaluate the cost-effectiveness of surveillance.
We included 714 patients with long-segment BO in a multicentre prospective cohort study and used a multistate Markov model to calculate progression rates from no dysplasia (ND) to low-grade dysplasia (LGD), high-grade dysplasia (HGD) and OAC. Progression rates were incorporated in a decision-analytic model, including costs and quality of life data. We evaluated different surveillance intervals for ND and LGD, endoscopic mucosal resection (EMR), radiofrequency ablation (RFA) and oesophagectomy for HGD or early OAC and oesophagectomy for advanced OAC. The incremental cost-effectiveness ratio (ICER) was calculated in costs per quality-adjusted life-year (QALY).
The annual progression rate was 2% for ND to LGD, 4% for LGD to HGD or early OAC and 25% for HGD or early OAC to advanced OAC. Surveillance every 5 or 4 years with RFA for HGD or early OAC and oesophagectomy for advanced OAC had ICERs of 5.283 and 62.619 per QALY for ND. Surveillance every five to one year had ICERs of 4.922, 30.067, 32.531, 41.499 and 75.601 per QALY for LGD. EMR prior to RFA was slightly more expensive, but important for tumour staging.
Based on a Dutch healthcare perspective and assuming a willingness-to-pay threshold of 35.000 per QALY, surveillance with EMR and RFA for HGD or early OAC, and oesophagectomy for advanced OAC is cost-effective every 5 years for ND and every 3 years for LGD.
Colonic fermentation in patients with UC in remission was compared with that in matched healthy subjects on habitual diets and when dietary fibre was increased.
Fibre intake, faecal output of fibre (measured as non-starch polysaccharide (NSP)), starch, microbiota and fermentation products, and whole gut transit time (WGTT) were assessed in association with habitual diet and when dietary intake of wheat bran (WB)-associated fibre and high amylose-associated resistant starch (RS) was increased in an 8-week, randomised, single-blind, cross-over study.
Despite a tendency to lower habitual fibre intake in UC patients, faecal NSP and starch concentrations were threefold higher than in controls, whereas concentrations of phenols and short-chain fatty acids, pH and WGTT were similar. Increasing RS/WB intake was well tolerated. In controls (n=10), it more than doubled faecal NSP and starch excretion (p=0.002 for both), had no effect on NSP usage and reduced WGTT (p=0.024). In UC patients (n=19), high intake of RS/WB tended to normalise gut transit, but did not increase the proportion of NSP fermented. Increasing intake of RS/WB had little effect on faecal fermentation patterns or the structure of the microbiota. However, faeces from the UC cohort had lower proportions of Akkermansia muciniphila and increased diversity within Clostridium cluster XIVa compared to controls.
Gut fermentation of NSP and starch is diminished in patients with UC. This cannot be explained by abnormal gut transit and was not corrected by increasing RS/WB intake, and may be due to abnormal functioning of the gut microbiota.
Australian New Zealand Clinical Trials Registry: ACTRN12614000271606.
Pancreatic acinar cell maturation is dependent on the activity of the pancreas transcription factor 1 (PTF1) complex. Induction of pancreatitis leads to MAP kinase activation and transient suppression of the acinar differentiation programme. We investigated the role of MAP kinase-interacting kinase 1 (Mnk1) in mouse exocrine pancreas development and in the response to secretagogue-induced pancreatitis.
Mnk1 expression was analysed using immunohistochemistry, RT-qPCR and western blotting. Ptf1a binding to Mnk1 was assessed by chromatin immunoprecipitation and qPCR. Acute pancreatitis was induced in wild type and Mnk1–/– mice by 7 h intraperitoneal injections of caerulein. In vitro amylase secretion and trypsinogen activation were assessed using freshly isolated acinar cells. In vivo secretion was quantified by secretin-stimulated MRI.
Mnk1 is expressed at the highest levels in pancreatic acinar cells and is a direct PTF1 target. Mnk1 is activated upon induction of pancreatitis and is indispensable for eIF4E phosphorylation. The pancreas of Mnk1–/– mice is histologically normal. Digestive enzyme content is significantly increased and c-Myc and Ccnd1 levels are reduced in Mnk1–/– mice. Upon induction of acute pancreatitis, Mnk1–/– mice show impaired eIF4E phosphorylation, activation of c-Myc and downregulation of zymogen content. Acinar cells show defective relocalisation of digestive enzymes, polarity defects and impaired secretory response in vitro and in vivo.
Mnk1 is a novel pancreatic acinar cell-specific stress response kinase that regulates digestive enzyme abundance and eIF4E phosphorylation. It is required for the physiological secretory response of acinar cells and for the homeostatic response to caerulein administration during acute pancreatitis.
Reported malignant progression rates for low-grade dysplasia (LGD) in Barrett's oesophagus (BO) vary widely. Expert histological review of LGD is advised, but limited data are available on its clinical value. This retrospective cohort study aimed to determine the value of an expert pathology panel organised in the Dutch Barrett's Advisory Committee (BAC) by investigating the incidence rates of high-grade dysplasia (HGD) and oesophageal adenocarcinoma (OAC) after expert histological review of LGD.
We included all BO cases referred to the BAC for histological review of LGD diagnosed between 2000 and 2011. The diagnosis of the expert panel was related to the histological outcome during endoscopic follow-up. Primary endpoint was development of HGD or OAC.
293 LGD patients (76% men; mean 63 years±11.9) were included. Following histological review, 73% was downstaged to non-dysplastic BO (NDBO) or indefinite for dysplasia (IND). In 27% the initial LGD diagnosis was confirmed. Endoscopic follow-up was performed in 264 patients (90%) with a median follow-up of 39 months (IQR 16–72). For confirmed LGD, the risk of HGD/OAC was 9.1% per patient-year. Patients downstaged to NDBO or IND had a malignant progression risk of 0.6% and 0.9% per patient-year, respectively.
Confirmed LGD in BO has a markedly increased risk of malignant progression. However, the vast majority of patients with community LGD will be downstaged after expert review and have a low progression risk. Therefore, all BO patients with LGD should undergo expert histological review of the diagnosis for adequate risk stratification.
I read with interest the study by Steenholdt et al
The study methodology has a number of questions that require attention.
First, 80% of the patients recruited for the study had non-fistulising disease, and the study entry criterion for them was having a Crohn's Disease Activity Index (CDAI) score of ≥220. Furthermore, the endpoint for clinical response for them was defined as CDAI reduction of ≥70 points. CDAI scores are largely driven by symptom reporting
Second, among the 33 patients who were randomised...]]>
Serum lipase activities above the threefold upper reference limit indicate acute pancreatitis. We investigated whether high lipase activity—within the reference range and in the absence of pancreatitis—are associated with genetic single nucleotide polymorphisms (SNP), and whether these identified SNPs are also associated with clinical pancreatitis.
Genome-wide association studies (GWAS) on phenotypes 'serum lipase activity' and 'high serum lipase activity' were conducted including 3966 German volunteers from the population-based Study-of-Health-in-Pomerania (SHIP). Lead SNPs associated on a genome-wide significance level were replicated in two cohorts, 1444 blood donors and 1042 pancreatitis patients.
Initial discovery GWAS detected SNPs within or near genes encoding the ABO blood group specifying transferases A/B (ABO), Fucosyltransferase-2 (FUT2), and Chymotrypsinogen-B2 (CTRB2), to be significantly associated with lipase activity levels in asymptomatic subjects. Replication analyses in blood donors confirmed the association of FUT-2 non-secretor status (OR=1.49; p=0.012) and ABO blood-type-B (OR=2.48; p=7.29x10–8) with high lipase activity levels. In pancreatitis patients, significant associations were found for FUT-2 non-secretor status (OR=1.53; p=8.56x10–4) and ABO-B (OR=1.69, p=1.0x10–4) with chronic pancreatitis, but not with acute pancreatitis. Conversely, carriers of blood group O were less frequently affected by chronic pancreatitis (OR=0.62; p=1.22x10–05) and less likely to have high lipase activity levels (OR=0.59; p=8.14x10–05).
These are the first results indicating that ABO blood type-B as well as FUT2 non-secretor status are common population-wide risk factors for developing chronic pancreatitis. They also imply that, even within the reference range, elevated lipase activities may indicate subclinical pancreatic injury in asymptomatic subjects.
A low FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols) diet reduces symptoms of IBS, but reduction of potential prebiotic and fermentative effects might adversely affect the colonic microenvironment. The effects of a low FODMAP diet with a typical Australian diet on biomarkers of colonic health were compared in a single-blinded, randomised, cross-over trial.
Twenty-seven IBS and six healthy subjects were randomly allocated one of two 21-day provided diets, differing only in FODMAP content (mean (95% CI) low 3.05 (1.86 to 4.25) g/day vs Australian 23.7 (16.9 to 30.6) g/day), and then crossed over to the other diet with ≥21-day washout period. Faeces passed over a 5-day run-in on their habitual diet and from day 17 to day 21 of the interventional diets were pooled, and pH, short-chain fatty acid concentrations and bacterial abundance and diversity were assessed.
Faecal indices were similar in IBS and healthy subjects during habitual diets. The low FODMAP diet was associated with higher faecal pH (7.37 (7.23 to 7.51) vs 7.16 (7.02 to 7.30); p=0.001), similar short-chain fatty acid concentrations, greater microbial diversity and reduced total bacterial abundance (9.63 (9.53 to 9.73) vs 9.83 (9.72 to 9.93) log10 copies/g; p<0.001) compared with the Australian diet. To indicate direction of change, in comparison with the habitual diet the low FODMAP diet reduced total bacterial abundance and the typical Australian diet increased relative abundance for butyrate-producing Clostridium cluster XIVa (median ratio 6.62; p<0.001) and mucus-associated Akkermansia muciniphila (19.3; p<0.001), and reduced Ruminococcus torques.
Diets differing in FODMAP content have marked effects on gut microbiota composition. The implications of long-term reduction of intake of FODMAPs require elucidation.
Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release.
mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators.
Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes.
Bone morphogenetic protein 6 (BMP6) has been identified as crucial regulator of iron homeostasis. However, its further role in liver pathology including non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH) is elusive. The aim of this study was to investigate the expression and function of BMP6 in chronic liver disease.
BMP6 was analysed in hepatic samples from murine models of chronic liver injury and patients with chronic liver diseases. Furthermore, a tissue microarray comprising 110 human liver tissues with different degree of steatosis and inflammation was assessed. BMP6-deficient (BMP6–/–) and wild-type mice were compared in two dietary NASH-models, that is, methionine choline-deficient (MCD) and high-fat (HF) diets.
BMP6 was solely upregulated in NAFLD but not in other murine liver injury models or diseased human livers. In NAFLD, BMP6 expression correlated with hepatic steatosis but not with inflammation or hepatocellular damage. Also, in vitro cellular lipid accumulation in primary human hepatocytes induced increased BMP6 expression. MCD and HF diets caused more hepatic inflammation and fibrosis in BMP6–/– compared with wild-type mice. However, only in the MCD and not in the HF diet model BMP6–/– mice developed marked hepatic iron overload, suggesting that further mechanisms are responsible for protective BMP6 effect. In vitro analysis revealed that recombinant BMP6 inhibited the activation of hepatic stellate cells (HSCs) and reduced proinflammatory and profibrogenic gene expression in already activated HSCs.
Steatosis-induced upregulation of BMP6 in NAFLD is hepatoprotective. Induction of BMP6-signalling may be a promising antifibrogenic strategy.
Hepatocellular carcinoma (HCC) risk-scores may predict HCC in Asian entecavir (ETV)-treated patients. We aimed to study risk factors and performance of risk scores during ETV treatment in an ethnically diverse Western population.
We studied all HBV monoinfected patients treated with ETV from 11 European referral centres within the VIRGIL Network.
A total of 744 patients were included; 42% Caucasian, 29% Asian, 19% other, 10% unknown. At baseline, 164 patients (22%) had cirrhosis. During a median follow-up of 167 (IQR 82–212) weeks, 14 patients developed HCC of whom nine (64%) had cirrhosis at baseline. The 5-year cumulative incidence rate of HCC was 2.1% for non-cirrhotic and 10.9% for cirrhotic patients (p<0.001). HCC incidence was higher in older patients (p<0.001) and patients with lower baseline platelet counts (p=0.02). Twelve patients who developed HCC achieved virologic response (HBV DNA <80 IU/mL) before HCC. At baseline, higher CU-HCC and GAG-HCC, but not REACH-B scores were associated with development of HCC. Discriminatory performance of HCC risk scores was low, with sensitivity ranging from 18% to 73%, and c-statistics from 0.71 to 0.85. Performance was further reduced in Caucasians with c-statistics from 0.54 to 0.74. Predicted risk of HCC based on risk-scores declined during ETV therapy (all p<0.001), but predictive performances after 1 year were comparable to those at baseline.
Cumulative incidence of HCC is low in patients treated with ETV, but ETV does not eliminate the risk of HCC. Discriminatory performance of HCC risk scores was limited, particularly in Caucasians, at baseline and during therapy.
We aimed to better clarify the role of germline variants of the FCG2 receptor, FCGR2A-H131R and FCGR3A-V158F, on the therapeutic efficacy of cetuximab in metastatic colorectal cancer (mCRC). A large cohort with sufficient statistical power was assembled.
To show a HR advantage of 0.6 in progression-free survival (PFS) for FCGR2A-HH versus the rest and FCGR3A-VV versus the rest, with an 80% power, 80 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) wild-type (KRAS-WT) and 52 KRAS-WT patients are required, respectively. This leads to a total sample size of 952 and 619 patients, respectively. Samples were collected from 1123 mCRC patients from 15 European centres treated with cetuximab alone or in combination with chemotherapy. Fc gamma receptor (FCGR) status was centrally genotyped. Two additional externally genotyped series were included.
Incidences of FCGR2A-HH and FCGR3A-VV in KRAS-WT patients were 220/660 (33%) and 109/676 (16.1%) respectively. There was no difference in median PFS (mPFS) for KRAS-WT patients with FCGR2A-HH (22.0 weeks; 95% CI18.8 to 25.2) versus non-HH (22.0 weeks; 95% CI 19.4 to 24.6) or for FCGR3A-VV (16.4 weeks; 95% CI 13.0 to 19.8) versus non-VV (23 weeks; 95% CI 21.1 to 24.9) (p=0.06). Median overall survival, response rate and disease control rate assessments showed no benefit for either HH or VV.
No differences in mPFS were found between the FCGR polymorphisms HH and the others and VV versus the others in KRAS-WT mCRC patients refractory to irinotecan, oxaliplatin and 5-fluorouracil treated with cetuximab. We cannot confirm the effects of other IgG1 antibodies, which may be weaker than previously suggested. Other markers may be needed to study the actual host antibody response to cetuximab.
The soluble preligand assembly domain (PLAD) of tumour necrosis factor receptor 1 (TNFR1) interferes with receptor trimerisation to block downstream signalling, and mediates Th17 suppression. We explored the therapeutic potential of recombinant PLAD.Fc protein on a spontaneous experimental colitis.
A T-cell-specific BLIMP-1 knockout mouse model with mixed Th1/Th17 responses, resembling human Crohn's disease (CD) was established, and its colitogenic phenotype was characterised. Mice, 9 weeks old, were treated with PLAD.Fc protein at 5 mg/kg of body weight twice per week for 16 weeks, and presence of colitis was monitored by the appearance of diarrhoea, weight loss, and by histological colonic scoring. Activation status, cytokine profiles, and transcription factors in T cells were further analysed.
The colitogenic phenotype in BLIMP-1 knockout mice was alleviated when an interleukin (IL)-23 knockdown transgene was introduced, indicating a therapeutic potential by downregulating IL-23-Th17 axis in these knockout mice. In PLAD.Fc-treated group, the mouse body weight remained stable and only mild disease scores were revealed. The percentage of naive CD4 T cells was increased and that of effector/memory CD4 T cells was decreased after PLAD.Fc-treatment. Moreover, the levels of IFN-, IL-17, IL-21, IL-22, IL-23R, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α were diminished. Strikingly, Th2-associated cytokines (IL-4, IL-13 and IL-10) in sera, as well as percentages of Th2 cells, were increased in PLAD.Fc-treated mice. However, PLAD.Fc-mediated suppression of effector phenotypes in Th1/Th17 was abrogated after neutralising IL-10.
The Th2 cytokine milieu induced by PLAD.Fc rebalanced T-helper cell subsets and conferred a protection against colitis in BLIMP-1 knockout mice.
The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host–microbial interactions).
We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays.
We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli.
This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host–microbial interactions.
Foxp3+CD4+CD25+ regulatory T cells (Tregs) control immune responses, but their role in acute viral hepatitis remains elusive. Herein, we investigated alteration in the peripheral blood Treg population during acute hepatitis A (AHA) and its implication in the immune-mediated liver injury.
The study included 71 patients with AHA, and peripheral blood mononuclear cells (PBMCs) were isolated. The suppressive activity of Treg population was determined by assessing anti-CD3/CD28-stimulated proliferation of Treg-depleted and reconstituted PBMCs. Treg cell frequency, phenotype and apoptosis in PBMCs were analysed by flow cytometry.
The frequency of circulating Tregs was reduced during AHA. Moreover, the suppressive activity of the total Treg pool in the peripheral blood was attenuated during AHA. Treg frequency and suppressive activity of the Treg population inversely correlated with the serum alanine aminotransferase level. Fas was overexpressed on Tregs during AHA, suggesting their susceptibility to Fas-induced apoptosis. Indeed, increased apoptotic death was observed in Tregs of patients with AHA compared with healthy controls. In addition, agonistic anti-Fas treatment further increased apoptotic death of Tregs from patients with AHA. The decreased Treg frequency and Fas overexpression on Tregs were not observed in other acute liver diseases such as acute hepatitis B, acute hepatitis C and toxic/drug-induced hepatitis.
The size of the Treg pool was contracted during AHA, resulting from apoptosis of Tregs induced by a Fas-mediated mechanism. Decrease in Treg numbers led to reduced suppressive activity of the Treg pool and consequently resulted in severe liver injury during AHA.
Chronic hepatitis B infection is endemic in New Zealand and has high prevalence in New Zealand Māori. Previous longitudinal studies in populations with predominantly vertically acquired chronic hepatitis B have shown low spontaneous hepatitis B surface-antigen (HBsAg) seroclearance rates: 0.5–1.4% annually (mean age of clearance 48 years). We report the 28-year follow-up data on clinical and serological outcomes in indigenous New Zealand Māori with early horizontally acquired HBV.
In 1984, community seroprevalence study identified 572 HBsAg-positive individuals, followed for 28 years. Liver-related mortality and hepatocellular carcinoma (HCC) incidence were compared between these 572 HBV carriers and 1140 HBsAg-negative matched case-controls. Surviving HBsAg-positive individuals have been followed up in 2012 with clinical assessment, blood tests and liver transient elastography. Rates of hepatitis B e-antigen (HBeAg) and HBsAg seroconversion were determined.
After total 13 187.4 person-years follow-up, 15 HBsAg-positive patients have developed HCC compared with none of the HBsAg-negative controls (p<0.001). 12 HBsAg-positive patients died from liver-related causes compared with none in the controls (p<0.001). Spontaneous HBeAg-seroconversion occurred in 91% of HBeAg-positive patients. Spontaneous HBsAg loss occurred in 33% overall (annual clearance rate 1.34%), with higher rates at older ages (1.05% in patients<20 years at entry vs 4.3% per annum >40 years at entry, p<0.0001). Median ages of HBeAg loss and HBsAg loss were 23 years (range 6–66 years) and 40 years (range 4–80 years), respectively.
Horizontally transmitted HBV in Maori is similarly associated with increased risk of liver-related mortality and HCC compared with Chinese, although absolute incidence rates are lower. The rates of HBeAg and HBsAg loss are high, and occur at an earlier age than previously reported.
Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes.
This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3.
We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound.
Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome.
This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.
Wide-field endoscopic mucosal resection (WF-EMR) is an alternative to surgery for treatment of advanced colonic mucosal neoplasia up to 120 mm in size, but has been criticised for its potentially high recurrence rates. We aimed to quantify recurrence at 4 months (early) and 16 months (late) following successful WF-EMR and identify its risk factors and clinical significance.
Ongoing multicentre, prospective, intention-to-treat analysis of sessile or laterally spreading colonic lesions ≥20 mm in size referred for WF-EMR to seven academic endoscopy units. Surveillance colonoscopy (SC) was performed 4 months (SC1) and 16 months (SC2) after WF-EMR, with photographic documentation and biopsy of the scar.
1134 consecutive patients were enrolled when 1000 successful EMRs were achieved, of whom 799 have undergone SC1. 670 were normal. Early recurrent/residual adenoma was present in 128 (16.0%, 95% CI 13.6% to 18.7%). One case was unknown. The recurrent/residual adenoma was diminutive in 71.7% of cases. On multivariable analysis, risk factors were lesion size >40 mm, use of argon plasma coagulation and intraprocedural bleeding. Of 670 with normal SC1, 426 have undergone SC2, with late recurrence present in 17 cases (4.0%, 95% CI 2.4% to 6.2%). Overall, recurrent/residual adenoma was successfully treated endoscopically in 135 of 145 cases (93.1%, 95% CI 88.1% to 96.4%). If the initial EMR was deemed successful and did not contain submucosal invasion requiring surgery, 98.1% (95% CI 96.6% to 99.0%) were adenoma-free and had avoided surgery at 16 months following EMR.
Following colonic WF-EMR, early recurrent/residual adenoma occurs in 16%, and is usually unifocal and diminutive. Risk factors were identified. Late recurrence occurs in 4%. Overall, recurrence was managed endoscopically in 93% of cases. Recurrence is not a significant clinical problem following WF-EMR, as with strict colonoscopic surveillance, it can be managed endoscopically with high success rates.
Current diagnostic methods for gastro-oesophageal reflux disease (GORD) have moderate sensitivity/specificity and can be invasive and expensive. Pepsin detection in saliva has been proposed as an 'office-based' method for GORD diagnosis. The aims of this study were to establish normal values of salivary pepsin in healthy asymptomatic subjects and to determine its value to discriminate patients with reflux-related symptoms (GORD, hypersensitive oesophagus (HO)) from functional heartburn (FH).
100 asymptomatic controls and 111 patients with heartburn underwent MII-pH monitoring and simultaneous salivary pepsin determination on waking, after lunch and dinner. Cut-off value for pepsin positivity was 16 ng/mL. Patients were divided into GORD (increased acid exposure time (AET), n=58); HO (normal AET and + Symptom Association Probability (SAP), n=26) and FH (normal AET and—SAP, n=27).
1/3 of asymptomatic subjects had pepsin in saliva at low concentration (0(0–59)ng/mL). Patients with GORD and HO had higher prevalence and pepsin concentration than controls (HO, 237(52–311)ng/mL and GORD, 121(29–252)ng/mL)(p<0.05). Patients with FH had low prevalence and concentration of pepsin in saliva (0(0–40) ng/mL). A positive test had 78.6% sensitivity and 64.9% specificity for diagnosis of GORD+HO (likelihood ratio: 2.23). However, one positive sample with >210 ng/mL pepsin suggested presence of GORD+HO with 98.2% specificity (likelihood ratio: 25.1). Only 18/84 (21.4%) of GORD+HO patients had 3 negative samples.
In patients with symptoms suggestive of GORD, salivary pepsin testing may complement questionnaires to assist office-based diagnosis. This may lessen the use of unnecessary antireflux therapy and the need for further invasive and expensive diagnostic methods.
To evaluate molecular profiles in the small bowel (SB) mucosa proximal to the pouch in ulcerative colitis (UC) patients after pouch surgery.
Patients were prospectively recruited and stratified according to disease behaviour: normal pouch (NP), chronic pouchitis (CP), and Crohn's-like disease of the pouch (CLDP). Biopsies obtained from the pouch and the normal-appearing proximal SB (40 cm proximal to the anal verge) were compared to ileal biopsies from normal controls (NC). A histopathological score based on the degree of polymorphonuclear and mononuclear infiltrates was used to assess inflammation in the pouch and the proximal SB. Gene expression analysis was performed using microarrays, and validated by real-time PCR. Gene ontology and clustering were evaluated by bioinformatics.
Thirty-six subjects were recruited (age 18–71 years, 16 males). Histopathology scores demonstrated minimal differences in the normal-appearing proximal SB of all groups. Nonetheless, significant (fold change ≥2, corrected p [FDR] ≤ 0.05) molecular alterations in the proximal SB were detected in all groups (NP n=9; CP n=80; and CLDP n=230) compared with NC. The magnitude of DUOX2 alteration in the proximal SB was highest. An increase of 6.0, 9.8 and 21.7 folds in DUOX2 expression in NP, CP, CLDP, respectively was observed. This was followed by alterations in MMP1, SLC6A14 and PGC. Gene alterations in the proximal SB overlapped with alterations within the pouch (76% and 97% overlap in CP and CLDP, respectively). Gene ontology analysis in the proximal SB and pouch were comparable.
Significant gene expression alterations exist in an apparently unaffected proximal SB. Alterations in the pouch and the proximal SB were comparable, suggesting that inflammation may not be limited to the pouch, but that it extends to the proximal SB.
We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome.
The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls.
Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity.
miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.
Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear.
We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo.
TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α.
Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies.
Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far.
We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing.
IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3+ T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments.
Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC.
Eosinophilic oesophagitis (EoE) is presumed to be an isolated oesophageal disease; yet other allergic diseases associated with eosinophilic infiltration of target tissues, such as asthma and eczema, show perturbed functions of other sites that may be involved in the diathesis of allergy modulation.
To analyse small intestinal permeability in patients with active EoE and in a separate group of patients in remission.
Small bowel permeability was determined using a dual sugar method by calculating lactulose:mannitol (L:M) ratio in 17 patients who met consensus criteria for active EoE (>15 eos/HPF) and 8 patients in remission (<5 eos/HPF). Data from 28 healthy controls was used for comparison.
Patients with active EoE had significantly higher L:M ratios when compared to controls (0.045 vs. 0.033, p<0.001) and to EoE in remission (0.041 vs. 0.027, p<.001). There was no significant difference in L:M between the group with EoEin remission and healthy controls. The current data show that L:M ratio of 0.033 also provides a reasonable cut-off that defined the active EoE group compared to patients in remission. The main component explaining the change in L:M ratio was increased absorption (and excretion) of lactulose ((1601±106 ug) when compared to the EoE remission (969±91 ug) and control (1043±92 ug, p<.001) groups.
Small bowel permeability is overall increased in patients with active EoE, and is normal in patients with EoE in remission when compared to healthy controls. The role of the small bowel in active EoE deserves further investigation.
Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined.
We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis.
Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5neg or low cell population but was distinct from typical antral Lgr5high stem cells. Treatment with progastrin interconverts Lgr5neg or low CCK2R+ cells into Lgr5high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis.
CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.
Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication.
To identify recurrent somatic mutations with prognostic significance in patients with CRC.
Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases.
Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6–14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study.
The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.
Intestinal dysbiosis has been associated with coeliac disease (CD), but whether the alterations are cause or consequence of the disease is unknown. This study investigated whether the human leukocyte antigen (HLA)-DQ2 genotype is an independent factor influencing the early gut microbiota composition of healthy infants at family risk of CD.
As part of a larger prospective study, a subset (n=22) of exclusively breastfed and vaginally delivered infants with either high genetic risk (HLA-DQ2 carriers) or low genetic risk (non-HLA-DQ2/8 carriers) of developing CD were selected from a cohort of healthy infants with at least one first-degree relative with CD. Infant faecal microbiota was analysed by 16S rRNA gene pyrosequencing and real time quantitative PCR.
Infants with a high genetic risk had significantly higher proportions of Firmicutes and Proteobacteria and lower proportions of Actinobacteria compared with low-risk infants. At genus level, high-risk infants had significantly less Bifidobacterium and unclassified Bifidobacteriaceae proportions and more Corynebacterium, Gemella, Clostridium sensu stricto, unclassified Clostridiaceae, unclassified Enterobacteriaceae and Raoultella proportions. Quantitative real time PCR also revealed lower numbers of Bifidobacterium species in infants with high genetic risk than in those with low genetic risk.
In high-risk infants negative correlations were identified between Bifidobacterium species and several genera of Proteobacteria (Escherichia/Shigella) and Firmicutes (Clostridium).
The genotype of infants at family risk of developing CD, carrying the HLA-DQ2 haplotypes, influences the early gut microbiota composition. This finding suggests that a specific disease-biased host genotype may also select for the first gut colonisers and could contribute to determining disease risk.
Endothelial progenitor cells (EPCs) circulate with increased numbers in the peripheral blood of patients with highly-vascularised hepatocellular carcinoma (HCC) and contribute to angiogenesis and neovascularisation. We hypothesised that angiogenic EPCs, that is, colony forming unit-endothelial cells (CFU-ECs), and outgrowth EPCs, that is, endothelial colony-forming cells, may exert paracrine effects on the behaviours and metastatic capacities of human hepatoma cells.
Various molecular and functional approaches ranging from in vitro cell culture studies on molecular signalling to in vivo investigations on cell invasion and orthotropic transplantation models in mice and clinical specimens from patients with HCC were used.
Monocyte chemotactic protein-1 (MCP-1) was identified as a critical mediator released from CFU-ECs to contribute to the chemotaxis of Huh7 and Hep3B cells by inducing their microRNA-21 (miR-21) biogenesis through the C-C chemokine receptor-2/c-Jun N-terminal kinase/activator protein-1 signalling cascade. CFU-EC-induction of miR-21 in these cells activated their Rac1 and matrix metallopeptidase-9 by silencing Rho GTPase-activating protein-24 and tissue inhibitor of metalloproteinase-3, respectively, leading to increased cell mobility. MCP-1-induction of miR-21 induced epithelial-mesenchymal transformation of Huh7 cells in vitro and their intrahepatic metastatic capability in vivo. Moreover, increased numbers of MCP-1+ EPCs and their positive correlations with miR-21 induction and metastatic stages in human HCC were found.
Our results provide new insights into the complexity of EPC-HCC interactions and indicate that anticancer therapies targeting either the MCP-1 released from angiogenic EPCs or the miR-21 biogenesis in HCC cells may prevent the malignant progression of primary tumours.
One of the conundrums of chronic infection with the hepatitis B virus (HBV) is identifying those individual patients who are at risk for the development of the serious sequelae of cirrhosis and hepatocellular carcinoma (HCC). It has been estimated that 25–40% of patients with chronic hepatitis B (CHB) who acquire the virus early in life (perinatal or early horizontal transmission from an HBeAg-positive source) will eventually develop these serious and disastrous consequences.
No therapy for non-alcoholic steatohepatitis (NASH) has been approved so far. Roux-en-y gastric bypass (RYGB) is emerging as a therapeutic option, although its effect on NASH and related hepatic molecular pathways is unclear from human studies. We studied the effect of RYGB on pre-existent NASH and hepatic mitochondrial dysfunction—a key player in NASH pathogenesis—in a novel diet-induced mouse model nicely mimicking human disease.
C57BL/6J mice were fed a high-fat high-sucrose diet (HF-HSD).
HF-HSD led to early obesity, insulin resistance and hypercholesterolaemia. HF-HSD consistently induced NASH (steatosis, hepatocyte ballooning and inflammation) with fibrosis already after 12-week feeding. NASH was accompanied by hepatic mitochondrial dysfunction, characterised by decreased mitochondrial respiratory chain (MRC) complex I and IV activity, ATP depletion, ultrastructural abnormalities, together with higher 4-hydroxynonenal (HNE) levels, increased uncoupling protein 2 (UCP2) and tumour necrosis factor-α (TNF-α) mRNA and free cholesterol accumulation. In our model of NASH and acquired mitochondrial dysfunction, RYGB induced sustained weight loss, improved insulin resistance and inhibited progression of NASH, with a marked reversal of fibrosis. In parallel, RYGB preserved hepatic MRC complex I activity, restored ATP levels, limited HNE production and decreased TNF-α mRNA.
Progression of NASH and NASH-related hepatic mitochondrial dysfunction can be prevented by RYGB. RYGB preserves respiratory chain complex activity, thereby restoring energy output, probably by limiting the amount of oxidative stress and TNF-α. These data suggest that modulation of hepatic mitochondrial function contributes to the favourable effect of RYBG on established NASH.
Programmed death-ligand 1 (PD-L1) has been shown to negatively regulate immune responses via its interaction with PD-1 receptor. In this study, we investigated the effects of PD-L1-Fc treatment on intestinal inflammation using two murine models of inflammatory colitis induced by dextran sulfate sodium (DSS) and T-cell transfer.
The anti-colitis effect of adenovirus expressing Fc-conjugated PD-L1 (Ad/PD-L1-Fc) and recombinant PD-L1-Fc protein was evaluated in DSS-treated wild-type and Rag-1 knockout (KO) mice. We examined differentiation of T-helper cells, frequency of innate immune cells, and cytokine production by dendritic cells (DCs) in the colon from DSS-treated mice after PD-L1-Fc administration. In Rag-1 KO mice reconstituted with CD4 CD45RBhigh T cells, we assessed the treatment effect of PD-L1-Fc protein on the development of colitis.
Administration of Ad/PD-L1-Fc significantly ameliorated DSS-induced colitis, which was accompanied by diminished frequency of interleukin (IL)-17A-producing CD4 T cells and increased interferon--producing CD4 T cells in the colon of DSS-fed mice. The anti-colitic effect of PD-L1-Fc treatment was also observed in DSS-treated Rag-1 KO mice, indicating lymphoid cell independency. PD-L1-Fc modulated cytokine production by colonic DCs and the effect was dependent on PD-1 expression. Furthermore, PD-L1-Fc protein could significantly reduce the severity of colitis in CD4 CD45RBhigh T-cell-transferred Rag-1 KO mice.
Based on the protective effect of PD-L1-Fc against DSS-induced and T-cell-induced colitis, our results suggest that PD-1-mediated inhibitory signals have a crucial role in limiting the development of colonic inflammation. This implicates that PD-L1-Fc may provide a novel therapeutic approach to treat inflammatory bowel disease.
Hepatocellular carcinoma (HCC) is the most common liver cancer. We characterised HCC associated with infection compared with non-HBV-related HCC to understand interactions between viral and hepatocyte genomic alterations and their relationships with clinical features.
Frozen HBV (n=86) or non-HBV-related (n=90) HCC were collected in two French surgical departments. Viral characterisation was performed by sequencing HBS and HBX genes and quantifying HBV DNA and cccDNA. Nine genes were screened for somatic mutations and expression profiling of 37 genes involved in hepatocarcinogenesis was studied.
HBX revealed frequent non-sense, frameshift and deletions in tumours, suggesting an HBX inactivation selected in HCC. The number of viral copies was frequently lower in tumour than in non-tumour tissues (p=0.0005) and patients with low HBV copies in the non-tumour liver tissues presented additional risk factor (HCV, alcohol or non-alcoholic steato-hepatitis, p=0.006). P53 was the most frequently altered pathway in HBV-related HCC (47%, p=0.001). Furthermore, TP53 mutations were associated with shorter survival only in HBV-related HCC (p=0.02) whereas R249S mutations were identified exclusively in migrants. Compared with other aetiologies, HBV-HCC were more frequently classified in tumours subgroups with upregulation of genes involved in cell-cycle regulation and a progenitor phenotype. Finally, in HBV-related HCC, transcriptomic profiles were associated with specific gene mutations (HBX, TP53, IRF2, AXIN1 and CTNNB1).
Integrated genomic characterisation of HBV and non-HBV-related HCC emphasised the immense molecular diversity of HCC closely related to aetiologies that could impact clinical care of HCC patients.
Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect.
Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)—an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis.
Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice.
These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.
Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis.
Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease.
OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis.
OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.
Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC strains adhere to enterocytes via interaction between type 1 pili and CEACAM6 receptors abnormally expressed on CD ileal mucosa, leading to gut inflammation. We analysed whether epigenetic mechanisms are involved in the upregulation of CEACAM6 expression in intestinal epithelial cells (IECs).
Methylation of CEACAM6 promoter was analysed using bisulfite sequencing and site-specific methylation by SnapShot. pCpGfree reporter system was used to analyse CEACAM6 promoter activity. Transgenic mice expressing human CEACAM6 fed either standard food or a low-methyl diet (LMD) were orally challenged with 109 AIEC LF82. After 3 days, gut-associated AIEC and proinflammatory cytokines were quantified.
Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within HIF-1-responsive elements (HREs). Methylation levels of CpG within CEACAM6 promoter were inversely correlated with CEACAM6 expression in IEC expressing various levels of CEACAM6. We show the critical role of HRE methylation and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC. This was confirmed in transgenic mice expressing human CEACAM6 fed a LMD. LMD-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favouring AIEC colonisation and subsequent inflammation.
HRE hypomethylation in CEACAM6 promoter correlates with high expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in mouse model mimicking CD susceptibility.
Dear Sir,
We read with interest the article by Habegger et al
Epigenetic alterations accumulate in normal-appearing tissues of patients with cancer, producing an epigenetic field defect. Cross-sectional studies show that the degree of the defect may be associated with risk in some types of cancer, especially cancers associated with chronic inflammation.
To demonstrate, by a multicentre prospective cohort study, that the risk of metachronous gastric cancer after endoscopic resection (ER) can be predicted by assessment of the epigenetic field defect using methylation levels.
Patients with early gastric cancer, aged 40–80 years, who planned to have, or had undergone, ER, were enrolled at least 6 months after Helicobacter pylori infection discontinued. Methylation levels of three preselected genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy, and the primary endpoint was defined as detection of a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrolment.
Among 826 patients enrolled, 782 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1–2, 2–3 and ≥3 years after the enrolment, respectively. The highest quartile of the miR-124a-3 methylation level had a significant univariate HR (95% CI) (2.17 (1.07 to 4.41); p=0.032) and a multivariate-adjusted HR (2.30 (1.03 to 5.10); p=0.042) of developing authentic metachronous gastric cancers. Similar trends were seen for EMX1 and NKX6-1.
Assessment of the degree of an epigenetic field defect is a promising cancer risk marker that takes account of life history.
We read the paper by Bajor et al with interest.
We therefore congratulate the authors on conducting this study. The concept that underlying organic GI disease may explain symptoms compatible with IBS is not novel. The current gold standard for diagnosing IBS, the Rome III...]]>
Molecular-based companion diagnostic tests are being used with increasing frequency to predict their clinical response to various drugs, particularly for molecularly targeted drugs. However, invasive procedures are typically required to obtain tissues for this analysis. Circulating tumour cells (CTCs) are novel biomarkers that can be used for the prediction of disease progression and are also important surrogate sources of cancer cells. Because current CTC detection strategies mainly depend on epithelial cell-surface markers, the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs.
We developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan).
Our biological capturing system can image epithelial and mesenchymal tumour cells with telomerase activities as GFP-positive cells. After sorting, direct sequencing or mutation-specific PCR can precisely detect different mutations in KRAS, BRAF and KIT genes in epithelial, mesenchymal or epithelial–mesenchymal transition-induced CTCs, and in clinical blood samples from patients with colorectal cancer.
This fluorescence virus-guided viable CTC capturing method provides a non-invasive alternative to tissue biopsy or surgical resection of primary tumours for companion diagnostics.
Autonomic nervous system dysfunction has been implicated in visceral hypersensitivity. However, the specific contribution of the parasympathetic nervous system (PNS) is unclear. We aimed to determine whether physiological and pharmacological manipulation of parasympathetic tone influences the development of hypersensitivity in a validated model of acid-induced oesophageal pain.
Prior to, and following, a 30-min distal oesophageal infusion of 0.15 M hydrochloric acid, pain thresholds to electrical stimulation were determined in the proximal non-acid exposed oesophagus in healthy subjects. Validated sympathetic (skin conductance response) and parasympathetic (cardiac vagal tone) parameters were measured at baseline and continuously thereafter. In study 1, 55 subjects were randomised in a pragmatic blinded crossover design to receive deep breathing or un-paced breathing during acid infusion. In study 2, 32 subjects were randomised in a blinded, crossover design to receive intravenous atropine or placebo (saline) with deep breathing during acid infusion.
Study 1: Deep breathing increased cardiac vagal tone (2.1±2.3 vs –0.3±2.3, p=0.0006) with concomitant withdrawal of skin conductance response (–0.6±4.9 vs 3±4.8, p=0.03) in comparison with un-paced breathing. Deep breathing prevented the development of acid-induced oesophageal hypersensitivity in comparison with sham breathing (p=0.0001). Study 2: Atropine, in comparison with placebo, blocked the attenuating effect of deep breathing on the development of acid-induced oesophageal hypersensitivity (p=0.046).
The development of oesophageal hyperalgesia is prevented by physiologically increasing parasympathetic tone. This effect is pharmacologically blocked with atropine, providing evidence that the PNS influences the development of oesophageal pain hypersensitivity.
Gastric cancer (GC) remains difficult to cure due to heterogeneity in a clinical challenge and the molecular mechanisms underlying this disease are complex and not completely understood. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in GC, but the role of specific miRNAs involved in this disease remains elusive. We performed next generation sequencing (NGS)-based whole-transcriptome profiling to discover GC-specific miRNAs, followed by functional validation of results.
NGS-based miRNA profiles were generated in matched pairs of GCs and adjacent normal mucosa (NM). Quantitative RT-PCR validation of miR-29c expression was performed in 274 gastric tissues, which included two cohorts of matched GC and NM specimens. Functional validation of miR-29c and its gene targets was undertaken in cell lines, as well as K19-C2mE and K19-Wnt1/C2mE transgenic mice.
NGS analysis revealed four GC-specific miRNAs. Among these, miR-29c expression was significantly decreased in GC versus NM tissues (p<0.001). Ectopic expression of miR-29c mimics in GC cell lines resulted in reduced proliferation, adhesion, invasion and migration. High miR-29c expression suppressed xenograft tumour growth in nude mice. Direct interaction between miR-29c and its newly discovered target, ITGB1, was identified in cell lines and transgenic mice. MiR-29c expression demonstrated a stepwise decrease in wild type hyperplasia-dysplasia cascade in transgenic mice models of GC.
MiR-29c acts as a tumour suppressor in GC by directly targeting ITGB1. Loss of miR-29c expression is an early event in the initiation of gastric carcinogenesis and may serve as a diagnostic and therapeutic biomarker for patients with GC.
Data to support treatment algorithms in ambulatory paediatric UC are scarce. We aimed to explore the 1 year outcome in an inception cohort of paediatric UC patients and to identify early predictors of good outcome that might serve as short term treatment targets.
A chart review of 115 children with new onset UC was performed (age 11±4.1 years; 58 (50%) males; 86 (75%) extensive colitis; 70 (61%) moderate–severe disease; 63 (55%) received steroids at baseline). We assessed the Paediatric Ulcerative Colitis Activity Index (PUCAI) and laboratory variables at the time of diagnosis and at 3 months, and endoscopy at diagnosis.
The 3 month PUCAI was the strongest predictor of 1 year sustained steroid free remission (SSFR) (area under the receiver operating characteristic curve (AUROC)=0.7 (95% CI 0.6 to 0.8) and colectomy by 2 years (AUROC=0.75 (0.6 to 0.89)). SSFR was achieved in 9/54 (17%) children who had active disease (PUCAI ≥10) at 3 months (negative predictive value (NPV)=83%) and by 4/46 (8.6%) of those with a PUCAI score >10; (NPV=91%, positive predictive value=52%; p<0.001), implying that PUCAI >10 at 3 months has a probability of 9% for achieving SSFR versus 48% with a PUCAI value of ≤10. None of the variables at baseline was predictive of SSFR or colectomy (endoscopic severity, disease extent, age, PUCAI or C reactive protein/erythrocyte sedimentation rate/albumin/haemoglobin; all AUROC<0.6, p>0.05) but baseline PUCAI predicted subsequent acute severe colitis and the need for salvage medical therapy.
Completeness of the early response appears more important than baseline UC severity for predicting outcome in children, and supports using PUCAI<10 as a feasible treatment goal. Our data suggest that treatment escalation should be considered with a PUCAI value of ≥10 at 3 months.
There are obvious merits in HCV clearance for selected patients on RRT. Successful treatment should prevent or substantially reduce the risk for liver-related problems including hepatic decompensation and primary liver cancer. These problems are...]]>
Several transport proteins located at the canalicular membrane of hepatocytes are required to maintain the correct secretory function of the liver (
Antiangiogenic strategies have been proposed as a promising new approach for the therapy of portal hypertension and chronic liver disease. Pigment epithelium-derived factor (PEDF) is a powerful endogenous angiogenesis inhibitor whose role in portal hypertension remains unknown. Therefore, we aimed at determining the involvement of PEDF in cirrhotic portal hypertension and the therapeutic efficacy of its supplementation.
PEDF expression profiling and its relationship with vascular endothelial growth factor (VEGF), neovascularisation and fibrogenesis was determined in bile duct-ligated (BDL) rats and human cirrhotic livers. The ability of exogenous PEDF overexpression by adenovirus-mediated gene transfer (AdPEDF) to inhibit angiogenesis, fibrogenesis and portal pressure was also evaluated in BDL rats, following prevention and intervention trials.
PEDF was upregulated in cirrhotic human and BDL rat livers. PEDF and VEGF protein expression and localisation in mesentery and liver increased in parallel with portal hypertension progression, being closely linked in time and space with mesenteric neovascularisation and liver fibrogenesis in BDL rats. Furthermore, AdPEDF increased PEDF bioavailability in BDL rats, shifting the net balance in the local abundance of positive (VEGF) and negative (PEDF) angiogenesis drivers in favour of attenuation of portal hypertension-associated pathological neovascularisation. The antiangiogenic effects of AdPEDF targeted only pathological angiogenesis, without affecting normal vasculature, and were observed during early stages of disease. AdPEDF also significantly decreased liver fibrogenesis (through metalloproteinase upregulation), portosystemic collateralisation and portal pressure in BDL rats.
This study provides compelling experimental evidence indicating that PEDF could be a novel therapeutic agent worthy of assessment in portal hypertension and cirrhosis.
Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy.
Aiming to explore the role of entry inhibitors for future clinical development, we investigated the antiviral efficacy and toxicity of entry inhibitors in combination with DAAs or other host-targeting agents (HTAs). Screening a large series of combinations of entry inhibitors with DAAs or other HTAs, we uncovered novel combinations of antivirals for prevention and treatment of HCV infection.
Combinations of DAAs or HTAs and entry inhibitors including CD81-, scavenger receptor class B type I (SR-BI)- or claudin-1 (CLDN1)-specific antibodies or small-molecule inhibitors erlotinib and dasatinib were characterised by a marked and synergistic inhibition of HCV infection over a broad range of concentrations with undetectable toxicity in experimental designs for prevention and treatment both in cell culture models and in human liver-chimeric uPA/SCID mice.
Our results provide a rationale for the development of antiviral strategies combining entry inhibitors with DAAs or HTAs by taking advantage of synergy. The uncovered combinations provide perspectives for efficient strategies to prevent liver graft infection and novel interferon-free regimens.
Inflammation plays crucial roles in the pathogenesis of several chronic inflammatory disorders, including Crohn's disease (CD) and UC, the two major forms of IBD. The urokinase plasminogen activator receptor (uPAR) exerts pleiotropic functions over the course of both physiological and pathological processes. uPAR not only has a key role in fibrinolysis but also modulates the development of protective immunity. Additionally, uPAR supports extracellular matrix degradation and regulates cell migration, adhesion and proliferation, thus influencing the development of inflammatory and immune responses. This study aimed to evaluate the role of uPAR in the pathogenesis of IBD.
The functional role of uPAR was assessed in established experimental models of colitis. uPAR deficiency effects on cytokine release, polarisation and bacterial phagocytosis were analysed in colonic macrophages. uPAR expression was analysed in surgical specimens collected from normal subjects and patients with IBD.
In mice, uPAR expression is positively regulated as colitis progresses. uPAR-KO mice displayed severe inflammation compared with wild-type littermates, as indicated by clinical assessment, endoscopy and colon histology. The absence of uPAR led to an increased production of inflammatory cytokines by macrophages that showed an M1 polarisation and impaired phagocytosis. In human IBD, CD68+ macrophages derived from the inflamed mucosa expressed low levels of uPAR.
These findings point to uPAR as an essential component of intestinal macrophage functions and unravel a new potential target to control mucosal inflammation in IBD.
We read the paper by Garsed et al,
At a time when licensed treatment options for IBS-D are lacking, we would like to congratulate the authors for...]]>
Over 100 ulcerative colitis (UC) loci have been identified by genome-wide association studies (GWASs) primarily in Caucasians (CEUs). Many of them have weak effects on disease susceptibility, and the bulk of the heritability cannot be ascribed to these loci. Very little is known about the genetic background of UC in non-CEU groups. Here we report the first GWAS on UC in a genetically distinct north Indian (NI) population.
A genome-wide scan was performed on 700 cases and 761 controls. 18 single-nucleotide polymorphisms (SNPs) (p<5x10–5) were genotyped in an independent cohort of 733 cases and 1148 controls. A linear mixed model was used for case–control association tests.
Seven novel human leucocyte antigen (HLA)-independent SNPs from chromosome 6, located in 3.8-1, BAT2, MSH5, HSPA1L, SLC44A4, CFB and NOTCH4, exceeded p<5x10–8 in the combined analysis. To assess the independent biological contribution of such genes from the extended HLA region, we determined the percentage alternative pathway activity of complement factor B (CFB), the top novel hit. The activity was significantly different (p=0.01) between the different genotypes at rs12614 in UC cases. Transethnic comparisons revealed a shared contribution of a fraction of UC risk genes between NI and CEU populations, in addition to genetic heterogeneity.
This study shows varying contribution of the HLA region to UC in different populations. Different environmental exposures and the characteristic genetic structure of the HLA locus across ethnic groups collectively make it amenable to the discovery of causative alleles by transethnic resequencing. This may lead to an improved understanding of the molecular mechanisms underlying UC.
Following chronic liver injury or when hepatocyte proliferation is impaired, ductular reactions containing hepatic progenitor cells (HPCs) appear in the periportal regions and can regenerate the liver parenchyma. HPCs exist in a niche composed of myofibroblasts, macrophages and laminin matrix. Galectin-3 (Gal-3) is a β-galactoside-binding lectin that binds to laminin and is expressed in injured liver in mice and humans.
We examined the role of Gal-3 in HPC activation. HPC activation was studied following dietary induced hepatocellular (choline-deficient ethionine-supplemented diet) and biliary (3,5-diethoxycarbonyl-1,4-dihydrocollidine supplemented diet) injury in wild type and Gal-3(–/–) mice.
HPC proliferation was significantly reduced in Gal-3(–/–) mice. Gal-3(–/–) mice failed to form a HPC niche, with reduced laminin formation. HPCs isolated from wild type mice secrete Gal-3 which enhanced adhesion and proliferation of HPCs on laminin in an undifferentiated form. These effects were attenuated in Gal3(–/–) HPCs and in wild type HPCs treated with the Gal-3 inhibitor lactose. Gal-3(–/–) HPCs in vitro showed increased hepatocyte function and prematurely upregulated both biliary and hepatocyte differentiation markers and regulated cell cycle genes leading to arrest in G0/G1.
We conclude that Gal-3 is required for the undifferentiated expansion of HPCs in their niche in injured liver.
The off-treatment durability of nucleos(t)ide analogue therapy in Asian hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) and the role of hepatitis B surface antigen (HBsAg) levels in predicting off-treatment durability has not been well investigated.
Following Asia-Pacific Association for the Study of the Liver guidelines, entecavir was stopped in Asian HBeAg negative patients treated for ≥2 years with undetectable HBV DNA levels on ≥3 separate occasions 6 months apart before treatment cessation. HBsAg and HBV DNA levels were prospectively monitored every 6–12 weeks for 48 weeks. Entecavir was restarted if there was virologic relapse (defined as HBV DNA >2000 IU/mL).
184 patients (mean age 53.9 years, 67.9% male) were recruited. The cumulative rate of virologic relapse at 24 and 48 weeks was 74.2% and 91.4%, respectively. The median HBV DNA level at virologic relapse was 11 000 (range 2115 to >1.98x108) IU/mL. 42 (25.8%) patients had elevated alanine aminotransferase (median level 97 U/L, range 37–1058 U/L) during virologic relapse. Mean rate of off-treatment HBsAg decline was 0.018 (±0.456) log IU/mL/year. No patients cleared HBsAg. There was no correlation between off-treatment serial HBsAg and HBV DNA levels (r=–0.026, p=0.541). HBsAg levels at the time of entecavir commencement, entecavir cessation and the subsequent rate of HBsAg reduction were not associated with virologic relapse (all p>0.05).
Entecavir cessation in Asian HBeAg negative CHB resulted in high rates of virologic relapse, suggesting nucleos(t)ide analogue therapy should be continued indefinitely until the recognised treatment endpoint of HBsAg seroclearance.
Understanding the neural circuitry of placebo analgesia in the context of visceral pain is increasingly important given evidence of clinical benefit of placebo treatment in IBS. This functional MRI study addressed placebo analgesia in IBS, UC and healthy control (HC) volunteers.
Painful rectal distensions were delivered in N=17 patients with IBS , N=15 patients with UC in remission, and sex-matched and age-matched HCs in an adaptation phase followed by intravenous application of saline combined with either positive instructions of pain relief (placebo) or neutral instructions (control). Neural activation during cued-pain anticipation and pain was analysed along with ratings of expected and perceived pain and measures of negative affectivity and salivary cortisol concentrations. Correlational analyses between placebo analgesia responses and negative affect were accomplished.
HC and UC revealed significant pain inhibition during placebo analgesia, as evidenced by reduced neural activation in pain-related brain areas. In contrast, patients with IBS failed to effectively engage neural downregulation of pain, as evidenced by the absence of placebo-induced changes in distension-induced brain activation, resulting in a significant group difference in the cingulate cortex compared with HC. Depression scores correlated with weaker placebo analgesia, whereas state and trait anxiety were not associated.
Patients with IBS failed to effectively engage neural downregulation of rectal distension-induced pain during placebo analgesia, indicating a specific deficit in cognitive pain inhibition, which may in part be mediated by depression.
Although paediatric-onset IBD is becoming more common, few medications have a registered paediatric indication. There are multiple hurdles to performing clinical trials in children, emphasising the importance of choosing an appropriate outcome measure, which can facilitate enrolment, and thereby also drug approval. The aim of this consensus statement is to highlight paediatric specific issues and key factors critical for the optimal conduct of paediatric IBD trials.
The Paediatric European Crohn's and Colitis Organisation (ECCO) committee has established an international expert panel to determine the best outcome measures in paediatric IBD, following a literature search and a modified Delphi process. All recommendations were endorsed by at least 80% agreement.
Recognising the importance of mucosal healing (MH), the panel defined steroid-free MH as primary outcome measure for all drugs of new category with one or two postintervention endoscopies per trial (at 8–12 weeks and/or 54 weeks). Since endoscopic evaluation is a barrier for recruitment in children, trials with medications already shown to induce MH in children or adults, could use paediatric-specific disease activity scores as primary outcome, including a modified Paediatric Crohn's Disease Activity Index in Crohn's disease and the Paediatric Ulcerative Colitis Activity Index in UC. Secondary outcomes should include safety issues, MR enterography-based damage and inflammatory scores (in Crohn's disease), faecal calprotectin, quality of life scales, and a patient-reported outcome.
It is crucial to perform paediatric trials early in the development of new drugs in order to reduce off-label use of IBD medication in children. The thoughtful choice of feasible and standardised outcome measures can help move us towards this goal.
Reliable tools to predict long-term outcome among patients with well compensated advanced liver disease due to chronic HCV infection are lacking.
Risk scores for mortality and for cirrhosis-related complications were constructed with Cox regression analysis in a derivation cohort and evaluated in a validation cohort, both including patients with chronic HCV infection and advanced fibrosis.
In the derivation cohort, 100/405 patients died during a median 8.1 (IQR 5.7–11.1) years of follow-up. Multivariate Cox analyses showed age (HR=1.06, 95% CI 1.04 to 1.09, p<0.001), male sex (HR=1.91, 95% CI 1.10 to 3.29, p=0.021), platelet count (HR=0.91, 95% CI 0.87 to 0.95, p<0.001) and log10 aspartate aminotransferase/alanine aminotransferase ratio (HR=1.30, 95% CI 1.12 to 1.51, p=0.001) were independently associated with mortality (C statistic=0.78, 95% CI 0.72 to 0.83). In the validation cohort, 58/296 patients with cirrhosis died during a median of 6.6 (IQR 4.4–9.0) years. Among patients with estimated 5-year mortality risks <5%, 5–10% and >10%, the observed 5-year mortality rates in the derivation cohort and validation cohort were 0.9% (95% CI 0.0 to 2.7) and 2.6% (95% CI 0.0 to 6.1), 8.1% (95% CI 1.8 to 14.4) and 8.0% (95% CI 1.3 to 14.7), 21.8% (95% CI 13.2 to 30.4) and 20.9% (95% CI 13.6 to 28.1), respectively (C statistic in validation cohort = 0.76, 95% CI 0.69 to 0.83). The risk score for cirrhosis-related complications also incorporated HCV genotype (C statistic = 0.80, 95% CI 0.76 to 0.83 in the derivation cohort; and 0.74, 95% CI 0.68 to 0.79 in the validation cohort).
Prognosis of patients with chronic HCV infection and compensated advanced liver disease can be accurately assessed with risk scores including readily available objective clinical parameters.
Impaired adaptive response to oxidative injuries is a fundamental mechanism central to the pathogenesis of chronic hepatitis C (CHC). Glycogen synthase kinase (GSK) 3β is an indispensable regulator of the oxidative stress response. However, the exact role of GSK3β in CHC is uncertain and was examined.
GSK3β and Nrf2 signalling pathways were examined in JFH1 HCV infected Huh7.5.1 hepatocytes, and also in liver biopsy specimens from CHC patients.
HCV infection elicited prominent Nrf2 antioxidant response in hepatocytes, marked by elevated expression of the Nrf2-dependent molecule haem oxygenase-1 and subsequent protection from apoptotic cell death. Inhibitory phosphorylation of GSK3β seems to be essential and sufficient for HCV-induced Nrf2 response. Mechanistically, GSK3β associated and physically interacted with Nrf2 in hepatocytes. In silico analysis revealed that Nrf2 encompasses multiple GSK3β phosphorylation consensus motifs, denoting Nrf2 as a cognate substrate of GSK3β. In the presence of TGFβ1, the HCV-induced GSK3β phosphorylation was blunted via a protein phosphatase 1-dependent mechanism and the cytoprotective Nrf2 response drastically impaired. This effect was counteracted by lithium, a selective inhibitor of GSK3β. In liver biopsy specimens from CHC patients, the expression of phosphorylated GSK3β positively correlated with Nrf2 expression and was inversely associated with the degree of liver injury. Moreover, CHC patients who received long-term lithium carbonate therapy primarily for concomitant psychiatric disorders exhibited much less liver injury, associated with enhanced hepatic expression of Nrf2.
Inhibition of GSK3β exerts hepatoprotection in CHC possibly through its direct regulation of Nrf2 antioxidant response.
Bacteria play a role in the onset and perpetuation of intestinal inflammation in IBD. Compositional alterations may also change the metabolic capacities of the gut bacteria.
To examine the metabolic activity of the microbiota of patients with Crohn's disease (CD), UC or pouchitis compared with healthy controls (HC) and determine whether eventual differences might be related to the pathogenesis of the disease.
Faecal samples were obtained from 40 HC, 83 patients with CD, 68 with UC and 13 with pouchitis. Disease activity was assessed in CD using the Harvey–Bradshaw Index, in UC using the UC Disease Activity Index and in pouchitis using the Pouchitis Disease Activity Index. Metabolite profiles were analysed using gas chromatography–mass spectrometry.
The number of metabolites identified in HC (54) was significantly higher than in patients with CD (44, p<0.001), UC (47, p=0.042) and pouchitis (43, p=0.036). Multivariate discriminant analysis predicted HC, CD, UC and pouchitis group membership with high sensitivity and specificity. The levels of medium-chain fatty acids (MCFAs: pentanoate, hexanoate, heptanoate, octanoate and nonanoate), and of some protein fermentation metabolites, were significantly decreased in patients with CD, UC and pouchitis. Hexanoate levels were inversely correlated to disease activity in CD (correlation coefficient=–0.157, p=0.046), whereas a significant positive correlation was found between styrene levels and disease activity in UC (correlation coefficient=0.338, p=0.001).
Faecal metabolic profiling in patients with IBD relative to healthy controls identified MCFAs as important metabolic biomarkers of disease-related changes.
Antimicrobial peptides (AMP) provide protection from infection by pathogenic microorganisms and restrict bacterial growth at epithelial surfaces to maintain mucosal homeostasis. In addition, they exert a significant anti-inflammatory activity. Here we analysed the anatomical distribution and biological activity of an orally administered AMP in the context of bacterial infection and host–microbial homeostasis.
The anatomical distribution as well as antibacterial and anti-inflammatory activity of the endogenous AMP cryptdin 2 and the synthetic peptide Pep19-2.5 at the enteric mucosal surface were analysed by immunostaining, functional viability and stimulation assays, an oral Salmonella enterica subsp. enterica sv. Typhimurium (S. Typhimurium) model and comparative microbiota analysis.
Endogenous cryptdin 2 was found attached to bacteria of the enteric microbiota within the intestinal mucus layer. Similarly, the synthetic peptide Pep19-2.5 attached rapidly to bacterial cells, exhibited a marked affinity for the intestinal mucus layer in vivo, altered the structural organisation of endotoxin in a mucus matrix and demonstrated potent anti-inflammatory and antibacterial activity. Oral Pep19-2.5 administration induced significant changes in the composition of the enteric microbiota as determined by high-throughput 16S rDNA sequencing. This may have contributed to the only transient improvement of the clinical symptoms after oral infection with S. Typhimurium.
Our findings demonstrate the anti-inflammatory activity and mucus affinity of the synthetic AMP Pep19-2.5 and characterise the influence on microbiota composition and enteropathogen infection after oral administration.
To examine patterns of colorectal cancer (CRC) screening uptake over three biennial invitation rounds in the National Health Service (NHS) Bowel Cancer Screening Programme (BCSP) in England.
We analysed data from the BCSP's Southern Hub for individuals (n=62 099) aged 60–64 years at the time of first invitation to screening with a follow-up period that allowed for two further biennial invitations. Data on sex, age and a neighbourhood-level measure of socioeconomic deprivation were used in the analysis. Outcomes included uptake of guaiac-based faecal occult blood (gFOB) test screening, inadequate gFOB screening (≥1 test kit(s) returned but failed to complete further gFOB tests needed to reach a conclusive test result), test positivity, compliance with follow-up examinations (usually colonoscopy) and diagnostic outcomes.
Overall gFOB uptake was 57.4% in the first, 60.9% in the second and 66.2% in third biennial invitation round. This resulted in 70.1% of the initial cohort having responded at least once, 60.7% at least twice and 44.4% three times. Participation in the first round was strongly predictive of participation in the second round ('Previous Responders': 86.6% vs 'Previous Non-Responders': 23.1%). Participation in the third round was highest among 'Consistent Screeners' (94.5%), followed by 'Late Entrants' (78.0%), 'Dropouts' (59.8%) and 'Consistent Non-Responders' (14.6%). Socioeconomic inequalities in uptake were observed across the three rounds, but sex inequalities decreased over rounds. Inadequate gFOB screening was influenced by screening history and socioeconomic deprivation. Screening history was the only significant predictor of follow-up compliance.
Screening history is associated with overall gFOB uptake, inadequate gFOB screening and follow-up compliance. Socioeconomic deprivation is also consistently associated with lower gFOB uptake and inadequate gFOB screening. Improving regular screening among identified 'at-risk' groups is important for the effectiveness of CRC screening programmes.
Genome-wide association studies (GWAS) have identified genetic variants within multiple risk loci as predisposing to intestinal inflammatory diseases, including Crohn's disease, ulcerative colitis and coeliac disease. Most risk variants affect regulation of transcription, but a critical challenge is to identify which genes and which cell types these variants affect. We aimed to characterise whole transcriptomes for each common T lymphocyte subset resident within the gut mucosa, and use these to infer biological insights and highlight candidate genes of interest within GWAS risk loci.
We isolated the four major intestinal T cell populations from pinch biopsies from healthy subjects and generated transcriptomes for each. We computationally integrated these transcriptomes with GWAS data from immune-related diseases.
Robust, high quality transcriptomic data were generated from 1 ng of RNA from precisely sorted cell subsets. Gene expression patterns clearly differentiated intestinal T cells from counterparts in peripheral blood and revealed distinct signalling pathways for each intestinal T cell subset. Intestinal-specific T cell transcripts were enriched in GWAS risk loci for Crohn's disease, ulcerative colitis and coeliac disease, but also specific extraintestinal immune-mediated diseases, allowing prediction of novel candidate genes.
This is the first report of transcriptomes for minimally manipulated intestinal T lymphocyte subsets in humans. We have demonstrated that careful processing of mucosal biopsies allows the generation of transcriptomes from as few as 1000 highly purified cells with minimal interindividual variation. Bioinformatic integration of transcriptomic data with recent GWAS data identified specific candidate genes and cell types for inflammatory pathologies.
Stress in the endoplasmic reticulum (ER) leads to activation of the unfolded protein response (UPR). Xbp1, a key component of the UPR has recently been linked to the risk of developing oesophageal squamous cell carcinoma, suggesting an important role for the UPR in the oesophageal epithelium. Here we examined the role of ER stress and the UPR in oesophageal epithelial homoeostasis.
We examined the expression of components of the UPR in the oesophageal epithelium. We used a pharmacological approach and a genetic approach to examine the effects of ER stress in vivo in the mouse oesophagus. The oesophagus of these mice was examined using immunohistochemistry and real-time reverse transcription (RT)-PCR.
Components of the UPR were heterogeneously expressed in the basal layer of the epithelium. Induction of ER stress by 24-h treatment with thapsigargin resulted in depletion of proliferating cells in the basal layer of the oesophagus and induced differentiation. We next activated the UPR by inducible deletion of the major ER chaperone Grp78 in Ah1Cre-Rosa26-LacZ-Grp78–/– mice in which mutant cells could be traced by expression of LacZ. In these mice LacZ-positive mutant cells in the basal layer lost their proliferative capacity, migrated towards the oesophageal lumen and were replaced by LacZ-negative non-mutant cells. We observed no apoptosis in mutant cells.
These results show that ER stress induces epithelial differentiation in precursor cells in the oesophageal epithelium. This UPR induced differentiation may serve as a quality control mechanism that protects against oesophageal cancer development.
We read the article by Schnúr et al
Bhat et al
The α4β7 integrin monoclonal antibody vedolizumab is hypothesised to be gut selective. Effects of vedolizumab on immune responses to parenterally or enterally administered antigens were investigated.
In this randomised, double-blind, placebo-controlled, phase I trial, healthy participants received a single intravenous dose of vedolizumab 750 mg (n=64) or placebo (n=63). After 4 days, participants began intramuscular hepatitis B vaccine (HBV; days 4, 32, 60) and oral cholera vaccine (OCV; days 4, 18) regimens. The study was designed to demonstrate a 15% non-inferiority margin for the between-group difference in the primary end point: percentage of participants with HBV seroconversion at day 74 (serum hepatitis B surface antigen (HBs) antibody titre ≥10 IU/L). OCV seroconversion at day 74 (>4-fold increase in serum cholera toxin (CT) antibodies) was a secondary end point.
A total of 56 (90.3%) placebo-treated and 54 (88.5%) vedolizumab-treated participants responded to HBV. Geometric mean anti-HBs titres were similar for placebo (114.4 IU/L) and vedolizumab (129.6 IU/L) at day 74. A total of 60 (96.8%) placebo-treated and 52 (82.5%) vedolizumab-treated participants responded to OCV at day 74. Geometric mean anti-CT IgG levels were higher for placebo than for vedolizumab at day 74 (9210.08 vs 3007.8 ELISA Units (EU)/mL) and day 32 (11629.3 vs 1575.4 EU/mL). Anti-CT IgA results were similar. Adverse events were consistent with previous experience. One serious adverse event (spontaneous abortion) was reported for placebo.
Vedolizumab did not alter the response to parenterally administered antigens but reduced the response to oral antigens, demonstrating its gut-selective mechanism of action.
NCT Number: 01981616; EudraCT Number: 2011-001874-24.
Precore (PC) variant (G1896A) and basal core promoter (BCP) variant (A1762T/G1764A) of HBV are associated with risk of hepatocellular carcinoma in HBV carriers. However, little is known about their impact on the adverse outcomes of hepatitis B e antigen (HBeAg)-negative hepatitis and liver cirrhosis.
251 spontaneous HBeAg seroconverters who had genotype B or C infection and received a long-term follow-up were enrolled. PC and BCP mutants were determined qualitatively and quantitatively to correlate with these adverse outcomes. The findings were validated by an independent case–control study, which included 184 patients with biopsy-proven liver fibrosis stages.
In the longitudinal cohort study, BCP mutant and possibly PC wild type were associated with cirrhosis development, but not HBeAg-negative hepatitis. Multivariable analysis showed that only BCP mutant was an independent risk factor for cirrhosis development. Using quantitative analysis of BCP mutant, a higher proportion of BCP mutant, defined as a continuous variable, a dichotomous variable or an ordinal variable, was associated with a higher risk of cirrhosis. If we chose 45% of BCP mutant as the cut-off, the risk of cirrhosis was higher in patients with BCP mutant ≥45% compared to <45% in the longitudinal cohort; this finding was validated by the case–control study (adjusted OR: 2.81, 95% CI 1.40 to 5.67).
A higher proportion of BCP mutant increases the risk of liver cirrhosis development in HBV carriers with genotype B or C infection.
However, we disagree that our study is underpowered, which was confirmed by highly significant differences between the groups. As indicated in the manuscript, we calculated the minimum sample size and even included more subjects than statistically required. We also want to point out that our n-values are in the upper range for a study that combines functional and structural measurements of epithelial integrity.
Questions were raised about the absence of Helicobacter pylori (HP) in our participants. Ten years ago, the prevalence of HP in the FD patient population of Belgium was low (17%), and has decreased even further since then.
Data comparing the efficacy and safety of combination therapy with peginterferon plus low-dose ribavirin and peginterferon monotherapy in treatment-naive haemodialysis patients with hepatitis C virus genotype 2 (HCV-2) infection are limited.
In this randomised trial, 172 patients received 24 weeks of peginterferon alfa-2a 135 μg/week plus ribavirin 200 mg/day (n=86) or peginterferon alfa-2a 135 μg/week (n=86). The efficacy and safety endpoints were sustained virological response (SVR) rate and adverse event (AE)-related withdrawal rate.
Compared with monotherapy, combination therapy had a greater SVR rate (74% vs 44%, relative risk (RR): 1.68 [95% CI 1.29 to 2.20]; p<0.001). The beneficial effect of combination therapy was more pronounced in patients with baseline viral load ≥800 000 IU/mL than those with baseline viral load <800 000 IU/mL (RR: 3.08 [95% CI 1.80 to 5.29] vs RR: 1.11 [95% CI 0.83 to 1.45]; interaction p=0.001). Patients receiving combination therapy were more likely to have a haemoglobin level of <8.5 g/dL (70% vs 8%, risk difference (RD): 62% [95% CI 50% to 73%]; p<0.001) and required a higher dosage [mean: 13 417vs 6667 IU/week, p=0.027] of epoetin β to manage anaemia than those receiving monotherapy. The AE-related withdrawal rates were 6% and 3% in combination therapy and monotherapy groups, respectively (RD: 2% [95% CI –4% to 9%]).
In treatment-naive haemodialysis patients with HCV-2 infection, combination therapy with peginterferon plus low-dose ribavirin achieved a greater SVR rate than peginterferon monotherapy. Most haemodialysis patients can tolerate combination therapy.
ClinicalTrial.gov number, NCT00491244.
To characterise the relationship between simvastatin and risk of acute pancreatitis (AP).
We conducted a retrospective cohort study (2006–2012) on data from an integrated healthcare system in southern California. Exposure to simvastatin was calculated from time of initial dispensation until 60 days following prescription termination. AP cases were defined by ICD-9 CM 577.0 and serum lipase≥3 times normal. Patients were censored at death, last follow-up, and onset of AP or end-of-study. Incidence rate of pancreatitis among simvastatin users was compared with the adult reference population. Robust Poisson regression was used to generate risk ratio (RR) estimates for simvastatin use adjusted for age, gender, race/ethnicity, gallstone-related disorders, hypertriglyceridaemia, smoking and alcohol dependence. Analysis was repeated for atorvastatin.
Among 3 967 859 adult patients (median duration of follow-up of 3.4 years), 6399 developed an initial episode of AP. A total of 707 236 patients received simvastatin during the study period. Patients that received simvastatin were more likely to have gallstone-related disorders, alcohol dependence or hypertriglyceridaemia compared with the reference population. Nevertheless, risk of AP was significantly reduced with simvastatin use, crude incidence rate ratio 0.626 (95% CL 0.588, 0.668), p<0.0001. In multivariate analysis, simvastatin was independently associated with reduced risk of pancreatitis, adjusted RR 0.29 (95% CL 0.27, 0.31) after adjusting for age, gender, race/ethnicity, gallstone disorders, alcohol dependence, smoking and hypertriglyceridaemia. Similar results were noted with atorvastatin, adjusted RR 0.33(0.29, 0.38).
Use of simvastatin was independently associated with reduced risk of AP in this integrated healthcare setting. Similar findings for atorvastatin suggest a possible class effect.
Bile acids may play a role in the pathogenesis of IBS. We investigated the potential effects of bile acids entering the colon and its role in the symptom pattern in IBS.
We measured 75Se-labelled homocholic acid-taurine (75SeHCAT) retention, and serum levels of 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor (FGF) 19 in patients with IBS (n=141) and control subjects (75SeHCAT n=29; C4 and FGF19 n=435). In patients with IBS stool frequency and form, as well as GI symptom severity were registered, and in a proportion of patients colonic transit time and rectal sensitivity were measured (n=66). An 8-week open-label treatment with colestipol was offered to patients with 75SeHCAT <20%, and the effect of treatment was evaluated with IBS severity scoring system and adequate relief of IBS symptoms.
Compared with controls, patients with IBS had lower 75SeHCAT values (p=0.005), higher C4c levels (C4 corrected for cholesterol) (p<0.001), but similar FGF19 levels. Abnormal 75SeHCAT retention (<10%) was seen in 18% of patients, whereas 23% had elevated C4c levels. Patients with IBS with 75SeHCAT retention <10% had more frequent stools, accelerated colonic transit time, rectal hyposensitivity, a higher body mass index, higher C4c and lower FGF19 levels. Colestipol treatment improved IBS symptoms (IBS severity scoring system 220±109 vs 277±106; p<0.01), and 15/27 patients fulfilled criteria for treatment response (adequate relief ≥50% of weeks 5–8).
Increased colonic bile acid exposure influences bowel habit and colonic transit time in patients with IBS. A high response rate to open label treatment with colestipol supports this, but placebo-controlled studies are warranted.
Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied.
We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls.
In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56dim NK cells at baseline was positively associated with IFNα treatment outcome in the patients.
We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.
Endoscopic surveillance for Barrett's oesophagus (BO) is limited by sampling error and the subjectivity of diagnosing dysplasia. We aimed to compare a biomarker panel on minimal biopsies directed by autofluorescence imaging (AFI) with the standard surveillance protocol to derive an objective tool for dysplasia assessment.
We performed a cross-sectional prospective study in three tertiary referral centres. Patients with BO underwent high-resolution endoscopy followed by AFI-targeted biopsies. 157 patients completed the biopsy protocol. Aneuploidy/tetraploidy; 9p and 17p loss of heterozygosity; RUNX3, HPP1 and p16 methylation; p53 and cyclin A immunohistochemistry were assessed. Bootstrap resampling was used to select the best diagnostic biomarker panel for high-grade dysplasia (HGD) and early cancer (EC). This panel was validated in an independent cohort of 46 patients.
Aneuploidy, p53 immunohistochemistry and cyclin A had the strongest association with dysplasia in the per-biopsy analysis and, as a panel, had an area under the receiver operating characteristic curve of 0.97 (95% CI 0.95 to 0.99) for diagnosing HGD/EC. The diagnostic accuracy for HGD/EC of the three-biomarker panel from AFI+ areas was superior to AFI– areas (p<0.001). Compared with the standard protocol, this panel had equal sensitivity for HGD/EC, with a 4.5-fold reduction in the number of biopsies. In an independent cohort of patients, the panel had a sensitivity and specificity for HGD/EC of 100% and 85%, respectively.
A three-biomarker panel on a small number of AFI-targeted biopsies provides an accurate and objective diagnosis of dysplasia in BO. The clinical implications have to be studied further.
A recent study by Ward et al
A few points may suggest that 233 may be still too low to...]]>
Octamer transcription factor 1 (OCT1) was found to be expressed in intestinal metaplasia and gastric cancer (GC), but the exact roles of OCT1 in GC remain unclear. The objective of this study was to determine the functional and prognostic implications of OCT1 in GC.
Expression of OCT1 was examined in paired normal and cancerous gastric tissues and the prognostic significance of OCT1 was analysed by univariate and multivariate survival analyses. The functions of OCT1 on synbindin expression and extracellular signal-regulated kinase (ERK) phosphorylation were studied in vitro and in xenograft mouse models.
The OCT1 gene is recurrently amplified and upregulated in GC. OCT1 overexpression and amplification are associated with poor survival in patients with GC and the prognostic significance was confirmed by independent patient cohorts. Combining OCT1 overexpression with American Joint Committee on Cancer staging improved the prediction of survival in patients with GC. High expression of OCT1 associates with activation of the ERK mitogen-activated protein kinase signalling pathway in GC tissues. OCT1 functions by transactivating synbindin, which binds to ERK DEF domain and facilitates ERK phosphorylation by MEK. OCT1-synbindin signalling results in the activation of ERK substrates ELK1 and RSK, leading to increased cell proliferation and invasion. Immunofluorescent study of human GC tissue samples revealed strong association between OCT1 protein level and synbindin expression/ERK phosphorylation. Upregulation of OCT1 in mouse xenograft models induced synbindin expression and ERK activation, leading to accelerated tumour growth in vivo.
OCT1 is a driver of synbindin-mediated ERK signalling and a promising marker for the prognosis and molecular subtyping of GC.
Nevertheless, we find the information provided about the patient selection criteria to be insufficient, and we would like a complementary explanation of this matter.
The 15 patients with FD included in this study were all negative for Helicobacter pylori. The fulfilment of this requirement may partially account for the small sample, but we would be in a better position to judge this if we knew the mean prevalence of H pylori infection in the adult population of Belgium, how many patients in the...]]>
Endoscopic surveillance of Barrett's oesophagus (BO) provides an opportunity to detect early stage oesophageal adenocarcinoma (OAC). We sought to determine the proportion of OAC patients with a prior diagnosis of BO on a population basis and to evaluate the influence of a prior diagnosis of BO on survival, taking into account lead and length time biases.
A retrospective population-based study of all OAC patients in Northern Ireland between 2003 and 2008. A prior BO diagnosis was determined by linkage to the Northern Ireland BO register. Stage distribution at diagnosis and histological grade were compared between patients with and without a prior BO diagnosis. Overall survival, using Cox models, was compared between patients with and without a prior BO diagnosis. The effect of adjusting the survival differences for histological grade and estimates of lead and length time bias was assessed.
There were 716 OAC cases, 52 (7.3%) of whom had a prior BO diagnosis. Patients with a prior BO diagnosis had significantly lower tumour stage (44.2% vs 11.1% had stage 1 or 2 disease; p<0.001), a higher rate of surgical resection (50.0% vs 25.5%; p<0.001) and had a higher proportion of low/intermediate grade tumours (46.2% vs 26.5%; p=0.011). A prior BO diagnosis was associated with significantly better survival (HR for death 0.39; 95% CI 0.27 to 0.58), which was minimally influenced by adjustment for age, sex and tumour grade (adjusted HR 0.44; 95% CI 0.30 to 0.64). Correction for lead time bias attenuated but did not abolish the survival benefit (HR 0.65; 95% CI 0.45 to 0.95) and further adjustment for length time bias had little effect.
The proportion of OAC patients with a prior diagnosis of BO is low; however, prior identification of BO is associated with an improvement in survival in OAC patients.
Anti-tumour necrosis factor α (TNFα) therapy effectively induces and maintains remission in Crohn's disease (CD). Up to 40% of patients, however, fail to respond to anti-TNFα.
To identify the mechanisms underlying the persistence of mucosal lesions in patients who fail to respond to anti-TNFα therapy.
An observational study based on whole-genome transcriptional analysis was carried out using intestinal biopsy specimens from patients with CD receiving (n=12) or not (n=10) anti-TNFα therapy. The transcriptional signature of responders was compared with that of non-responders after anti-TNFα therapy. Controls with non-inflammatory bowel disease (non-IBD) (n=17) were used for comparisons. Genes of interest were validated by real-time RT-PCR in an independent cohort of patients with CD receiving (n=17) or not (n=16) anti-TNFα and non-IBD controls (n=7).
We confirmed that response to anti-TNFα is accompanied by significant regulation of a large number of genes, including IL1B, S100A8, CXCL1, which correlated with endoscopic activity. Remarkably, patients who failed to respond to anti-TNFα showed a mixed signature, maintaining increased expression of IL1B, IL17A and S100A8, while showing significant modulation of other genes commonly upregulated in active CD, including IL6 and IL23p19.
Our results show that anti-TNFα therapy significantly downregulates a subset of inflammatory genes even in patients who fail to achieve endoscopic remission, suggesting that these genes may not be dominant in driving inflammation in non-responders. On the other hand, we identified IL1B and IL17A as genes that remained altered in non-responders, pointing to potentially more relevant targets for modulating mucosal damage in refractory patients.
IBS aggregates in families, but the familial risk of IBS has only been determined in first-degree relatives and spouses. This nationwide study aimed to determine the familial risk of IBS in first-degree, second-degree, and third-degree relatives and spouses of affected individuals in order to estimate the relative influences of genes and shared family environment.
We performed a case-cohort study. The Swedish Multigeneration Register was linked to the Hospital Discharge Register for the period 1987–2010 and the Swedish Outpatient Care Register for 2001–2010. ORs for IBS were calculated for relatives of individuals who had been diagnosed with IBS compared with relatives of individuals unaffected by IBS as the reference group. ORs were also determined for IBS cases diagnosed in primary healthcare in four Swedish counties (2001–2007).
The ORs for IBS were 1.75 in siblings (95% CI 1.63 to 1.89), 1.82 in offspring (1.67 to 1.97), 1.90 in parents (1.76 to 2.05), 1.10 in maternal half-siblings (0.88 to 1.39), 1.78 in paternal half-siblings (1.48 to 2.15), 1.27 in nieces/nephews (1.18 to 1.38), 1.11 in cousins (1.04 to 1.18), and 1.51 in spouses (1.24 to 1.84) of probands diagnosed with IBS. The OR for probands diagnosed in primary healthcare was 1.82 in siblings (1.52 to 2.18), and 1.82 in offspring (1.49 to 2.21).
The increased IBS risk among first-degree relatives and also second-degree and third-degree relatives indicates a genetic component of the familial clustering of IBS. However, a non-genetic contribution is also suggested by the increased risk among spouses.
To identify novel hereditary CRC genes, we studied an Amsterdam I family (hereditary non-polyposis CRC) with no mutations in the DNA mismatch repair (MMR) genes (
Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) plays an important role in controlling the precisely regulated switch between gene repression and gene activation in transcriptional regulation. We investigated its biological function and clinical significance in esophageal squamous cell carcinoma (ESCC).
Immunoblotting and immunochemistry were used to determine TBL1XR1 expression in ESCC cell lines, ESCC clinical tissues and 230 clinicopathologically characterised ESCC specimens. The role of TBL1XR1 in lymphangiogenesis and lymphatic metastasis was examined by tube formation, cell invasion and wound-healing assays in vitro, and by a popliteal lymph node metastasis model in vivo. The molecular mechanism by which TBL1XR1 upregulates vascular endothelial growth factor C (VEGF-C) expression was explored using real-time PCR, ELISA, luciferase reporter assay and chromatin immunoprecipitation.
TBL1XR1 expression was significantly upregulated in ESCC, positively correlated with disease stage and patient survival, and identified as an independent prognostic factor for patient outcome. We found that TBL1XR1 overexpression promoted lymphangiogenesis and lymphatic metastasis in ESCC in vitro and in vivo, whereas TBL1XR1 silencing had the converse effect. We demonstrated that TBL1XR1 induced VEGF-C expression by binding to the VEGF-C promoter. We confirmed the correlation between TBL1XR1 and VEGF-C expression in a large cohort of clinical ESCC samples and through analysis of published datasets in gastric, colorectal and breast cancer.
Our results demonstrated that TBL1XR1 induced lymphangiogenesis and lymphatic metastasis in ESCC via upregulation of VEGF-C, and may represent a novel prognostic biomarker and therapeutic target for patients with ESCC.
Since the publication of the first Asia Pacific Consensus on Colorectal Cancer (CRC) in 2008, there are substantial advancements in the science and experience of implementing CRC screening. The Asia Pacific Working Group aimed to provide an updated set of consensus recommendations.
Members from 14 Asian regions gathered to seek consensus using other national and international guidelines, and recent relevant literature published from 2008 to 2013. A modified Delphi process was adopted to develop the statements.
Age range for CRC screening is defined as 50–75 years. Advancing age, male, family history of CRC, smoking and obesity are confirmed risk factors for CRC and advanced neoplasia. A risk-stratified scoring system is recommended for selecting high-risk patients for colonoscopy. Quantitative faecal immunochemical test (FIT) instead of guaiac-based faecal occult blood test (gFOBT) is preferred for average-risk subjects. Ancillary methods in colonoscopy, with the exception of chromoendoscopy, have not proven to be superior to high-definition white light endoscopy in identifying adenoma. Quality of colonoscopy should be upheld and quality assurance programme should be in place to audit every aspects of CRC screening. Serrated adenoma is recognised as a risk for interval cancer. There is no consensus on the recruitment of trained endoscopy nurses for CRC screening.
Based on recent data on CRC screening, an updated list of recommendations on CRC screening is prepared. These consensus statements will further enhance the implementation of CRC screening in the Asia Pacific region.
Capecitabine is an oral 5-fluorouracil (5-FU) pro-drug commonly used to treat colorectal carcinoma and other tumours. About 35% of patients experience dose-limiting toxicity. The few proven genetic biomarkers of 5-FU toxicity are rare variants and polymorphisms, respectively, at candidate loci dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS).
We investigated 1456 polymorphisms and rare coding variants near 25 candidate 5-FU pathway genes in 968 UK patients from the QUASAR2 clinical trial.
We identified the first common DPYD polymorphisms to be consistently associated with capecitabine toxicity, rs12132152 (toxicity allele frequency (TAF)=0.031, OR=3.83, p=4.31x10–6) and rs12022243 (TAF=0.196, OR=1.69, p=2.55x10–5). rs12132152 was particularly strongly associated with hand-foot syndrome (OR=6.1, p=3.6x10–8). The rs12132152 and rs12022243 associations were independent of each other and of previously reported DPYD toxicity variants. Next-generation sequencing additionally identified rare DPYD variant p.Ala551Thr in one patient with severe toxicity. Using functional predictions and published data, we assigned p.Ala551Thr as causal for toxicity. We found that polymorphism rs2612091, which lies within an intron of ENOSF1, was also associated with capecitabine toxicity (TAF=0.532, OR=1.59, p=5.28x10–6). ENSOF1 is adjacent to TYMS and there is a poorly characterised regulatory interaction between the two genes/proteins. Unexpectedly, rs2612091 fully explained the previously reported associations between capecitabine toxicity and the supposedly functional TYMS variants, 5'VNTR 2R/3R and 3'UTR 6 bp ins-del. rs2612091 genotypes were, moreover, consistently associated with ENOSF1 mRNA levels, but not with TYMS expression.
DPYD harbours rare and common capecitabine toxicity variants. The toxicity polymorphism in the TYMS region may actually act through ENOSF1.
To estimate risk of colorectal cancer (CRC) for first-degree relatives of CRC cases based on CRC molecular subtypes and tumour pathology features.
We studied a cohort of 33 496 first-degree relatives of 4853 incident invasive CRC cases (probands) who were recruited to the Colon Cancer Family Registry through population cancer registries in the USA, Canada and Australia. We categorised the first-degree relatives into four groups: 28 156 of 4095 mismatch repair (MMR)-proficient probands, 2302 of 301 MMR-deficient non-Lynch syndrome probands, 1799 of 271 suspected Lynch syndrome probands and 1239 of 186 Lynch syndrome probands. We compared CRC risk for first-degree relatives stratified by the absence or presence of specific tumour molecular pathology features in probands across each of these four groups and for all groups combined.
Compared with first-degree relatives of MMR-proficient CRC cases, a higher risk of CRC was estimated for first-degree relatives of CRC cases with suspected Lynch syndrome (HR 2.06, 95% CI 1.59 to 2.67) and with Lynch syndrome (HR 5.37, 95% CI 4.16 to 6.94), but not with MMR-deficient non-Lynch syndrome (HR 1.04, 95% CI 0.82 to 1.31). A greater risk of CRC was estimated for first-degree relatives if CRC cases were diagnosed before age 50 years, had proximal colon cancer or if their tumours had any of the following: expanding tumour margin, peritumoral lymphocytes, tumour-infiltrating lymphocytes or synchronous CRC.
Molecular pathology features are potentially useful to refine screening recommendations for first-degree relatives of CRC cases and to identify which cases are more likely to be caused by genetic or other familial factors.
Knowledge of the cellular mechanisms involved in homeostasis of human squamous oesophagus in the steady state and following chronic injury is limited. We aimed to better understand these mechanisms by using a functional 3D approach.
Proliferation, mitosis and the expression of progenitor lineage markers were assessed in normal squamous oesophagus from 10 patients by immunofluorescence on 3D epithelial whole mounts. Cells expressing differential levels of epithelial and progenitor markers were isolated using flow cytometry sorting and characterised by qPCR and IF. Their self-renewing potential was investigated by colony forming cells assays and in vitro organotypic culture models.
Proliferation and mitotic activity was highest in the interpapillary basal layer and decreased linearly towards the tip of the papilla (p<0.0001). The orientation of mitosis was random throughout the basal layer, and asymmetric divisions were not restricted to specific cell compartments. Cells sorted into distinct populations based on the expression of epithelial and progenitor cell markers (CD34 and EpCAM) showed no difference in self-renewal in 2D culture, either as whole populations or as single cells. In 3D organotypic cultures, all cell subtypes were able to recapitulate the architecture of the tissue of origin and the main factor determining the success of the 3D culture was the number of cells plated, rather than the cell type.
Oesophageal epithelial cells demonstrate remarkable plasticity for self-renewal. This situation could be viewed as an ex vivo wounding response and is compatible with recent findings in murine models.
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a potentially lethal autosomal recessive liver disease associated with mutations in ABCB4, the gene encoding the canalicular translocator of phosphatidylcholine MDR3. While some affected children benefit from ursodeoxycholic acid (UDCA) therapy, others evolve to end-stage liver disease. We aimed to evaluate whether these different outcomes are related to the impact of ABCB4 mutations.
Six children with PFIC3 were investigated by sequencing of ABCB4 exons and flanking intron-exon boundaries and by immunohistochemistry. ABCB4 missense mutations were phenotyped in vitro by assessing their effects on MDR3 expression, subcellular localisation, and phosphatidylcholine-translocating activity. The resulting data were contrasted with the clinical outcomes.
Eight distinct ABCB4 mutations were identified: one nonsense, one splicing and six missense mutations, four of which (G68R, T201M, P479L, D459H) affected MDR3 expression level. G68R and D459H also led to retention of the protein in endoplasmic reticulum. Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees. Three children with mutations that caused a total loss of MDR3 expression/function manifested progressive liver disease refractory to UDCA treatment. This was also the case in a patient carrying two different mutations that, in combination, resulted in a 90% reduction in total MDR3 activity. A favourable response to UDCA was achieved in two patients with estimated MDR3 activities of 50% and 33%, respectively.
These data provide experimental evidence of the correlation between the degree of MDR3 floppase activity and the clinical outcomes of PFIC3.
The genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD.
Exome sequencing and immunological profiling were performed in a patient with early onset Crohn's disease (CD). The coding region of the gene encoding X-linked inhibitor of apoptosis protein (XIAP) was sequenced in samples of 275 paediatric IBD and 1047 adult-onset CD patients. XIAP genotyping was performed in samples of 2680 IBD patients and 2864 healthy controls. Functional effects of the variants identified were investigated in primary cells and cultured cell lines.
Our results demonstrate the frequent occurrence of private variants in XIAP in about four percent of male patients with paediatric-onset CD. While XIAP mutations are known to be associated with the primary immunodeficiency (PID) X-linked lymphoproliferative disease type 2 (XLP2), CD patients described here exhibited intestinal inflammation in the absence of XLP2 and harboured a spectrum of mutations partially distinct from that observed in XLP2. The majority of XIAP variants identified was associated with a selective defect in NOD1/2 signalling, impaired NOD1/2-mediated activation of NF-B, and altered NF-B-dependent cytokine production.
This study reveals the unanticipated, frequent occurrence of XIAP variants in male paediatric-onset CD. The link between XIAP and NOD1/2, and the association of XIAP variants with XLP2, support the concept of PID in a subset of IBD patients. Moreover, these studies provide a rationale for the implementation of XIAP sequencing in clinical diagnostics in male patients with severe CD.
Liver tumour-initiating cells (T-ICs) are critical for hepatocarcinogenesis. However, the underlying mechanism regulating the function of liver T-ICs remains unclear.
Tissue microarrays containing 242 hepatocellular carcinoma (HCC) samples were used for prognostic analysis. Magnetically activated cell sorting was used to isolate epithelial cell adhesion molecule (EPCAM)-positive cells. The gene expressions affected by miR-429 were determined by arrays. Co-immunoprecipitation was used to study interactions among retinoblastoma protein (RB1), Rb binding protein 4 (RBBP4) and E2F transcription factor 1 (E2F1). The DNA methylation status in CpG islands was detected by quantitative methylation analysis. miRNAs in microvesicles were isolated by a syringe filter system.
The significant prognosis factor miR-429 was upregulated in HCC tissues and also in primary liver T-ICs isolated from clinical samples. The enrichment of miR-429 in EPCAM+ T-ICs contributed to hepatocyte self-renewal, malignant proliferation, chemoresistance and tumorigenicity. A novel functional axis involving miR-429, RBBP4, E2F1 and POU class 5 homeobox 1 (POU5F1 or OCT4) governing the regulation of liver EPCAM+ T-ICs was established in vitro and in vivo. The molecular mechanism regulating miR-429 expression, involving four abnormal hypomethylated sites upstream of the miR-200b/miR-200a/miR-429 cluster, was first defined in both EPCAM+ liver T-ICs and very early-stage HCC tissues. miR-429 secreted by high-expressing cells has the potential to become a proactive signalling molecule to mediate intercellular communication.
Epigenetic modification of miR-429 can manipulate liver T-ICs by targeting the RBBP4/E2F1/OCT4 axis. This miRNA might be targeted to inactivate T-ICs, thus providing a novel strategy for HCC prevention and treatment.
They stipulated that exclusion of patients with vitamin D deficiency limited the significance of the performed study, as this deficiency is frequently observed in Crohn's disease. It is however well known that replenishment of vitamin D (and calcium) in patients with vitamin D deficiency would lead to a rapid improvement of bone density, irrespective of any effect of risedronate. As assessment of a therapeutic effect of the latter was our primary study aim, we decided to exclude patients with vitamin D deficiency.
In addition to their observation that present-day treatment strategies for osteopenia in patients with Crohn's disease are primarily based on suboptimal study data, a recent meta-analysis from 19 randomised controlled trials concerning low bone mineral density in patients with IBD, though not including our...]]>
The antiviral efficacy of nucleos(t)ide analogues whose main limitation is relapse after discontinuation requires long-term therapy. To overcome the risk of relapse and virological breakthrough during long-term therapy, we performed a phase I/II, open, prospective, multicentre trial using a HBV envelope-expressing DNA vaccine.
70 patients treated effectively with nucleos(t)ide analogues for a median of 3 years (HBV DNA <12 IU/mL for at least 12 months) were randomised into two groups: one received five intramuscular injections of vaccine (weeks 0, 8, 16, 40 and 44) and one did not receive the vaccine. Analogues were stopped after an additional 48 weeks of treatment in patients who maintained HBV DNA <12 IU/mL with no clinical progression and monthly HBV DNA for 6 months. The primary endpoint was defined as viral reactivation at week 72 (HBV DNA >120 IU/mL) or impossibility of stopping treatment at week 48.
Reactivation occurred in 97% of each group after a median 28 days without liver failure but with an HBV DNA <2000 IU/mL in 33%; 99% of adverse reactions were mild to moderate. Immune responses were evaluated by enzyme-linked immunosorbent spot and proliferation assays: there was no difference in the percentage of patients with interferon- secreting cells and a specific T-cell proliferation to HBcAg but not to HBsAg after reactivation in each group.
Although it is fairly well tolerated, the HBV DNA vaccine does not decrease the risk of relapse in HBV-treated patients or the rate of virological breakthrough, and does not restore the anti-HBV immune response despite effective viral suppression by analogues.
We would like to respond to Dr Pariente's letter, which addresses our recently reported results regarding the seroclearance of HBV seromarkers and subsequent risk for hepatocellular carcinoma (HCC).
First, he suggests that there may be underestimation of the protective roles of marker seroclearance, since incidence rates of HCC were measured from baseline, and that the duration of seromarkers' positivity would be a better indicator of HCC risk. In order to truly measure the effect of HBV DNA and Hepatitis B surface Antigen (HBsAg) seroclearance on HCC incidence, we would need a large cohort of individuals followed up since birth for subsequent outcomes. However, this was not feasible in this study. Thus, left truncation that often occurs in cohort studies may have resulted in some underestimation of effect. However, the number of cases was not sufficient...]]>
We thank Dr Tøttrup and colleagues for their comments
Both authors contributed equally in this manuscript.
Both authors contributed equally in this manuscript.
]]> Not commissioned; internally peer reviewed.
Not commissioned; internally peer reviewed.
With great interest we have read the paper by Rungoe et al
This fact may induce some bias when interpreting the...]]>
Liu et al The incidence rates of HCC are measured from baseline (as the characteristics of the patients) and not from the first occurrence of seromarkers clearance, causing probably an underestimation of the protective roles of the clearances of HBsAg and HBV DNA against HCC occurrence. In other words, the predictive value of the duration of seromarkers positivity could be a better indicator of the risk of HCC that the seromarkers state at the end of the follow-up. The figure describing the HCC cumulative lifetime incidence is frightening, but is it the reality? As this figure does not take into account other important predictive factors of HCC, such as age at entry in the...]]>
The incidence rates of HCC are measured from baseline (as the characteristics of the patients) and not from the first occurrence of seromarkers clearance, causing probably an underestimation of the protective roles of the clearances of HBsAg and HBV DNA against HCC occurrence. In other words, the predictive value of the duration of seromarkers positivity could be a better indicator of the risk of HCC that the seromarkers state at the end of the follow-up.
The figure describing the HCC cumulative lifetime incidence is frightening, but is it the reality? As this figure does not take into account other important predictive factors of HCC, such as age at entry in the...]]>
We read with great interest van Bodegraven et al's
One of the exclusion criteria for patients in this study was vitamin D deficiency. Vitamin D deficiency has previously been noted to be common in patients with Crohn's disease.
We read with interest the two excellent studies on interval colorectal cancers (CRC) following colonoscopy and wish to present important data relevant to the prevention of interval CRC. In a pooled multicohort analysis, Robertson and coauthors found that invasive cancer was diagnosed in 0.6% of patients after clearing colonoscopy among eight large North American trials, and concluded that half of these were likely missed lesions.
We previously described that non-polypoid colorectal neoplasms (NP-CRN) harbour more advanced pathology than polypoid neoplasms.
Using available data, we summarised the learning curve (see
From question on page
Macroscopically, the excreted strand had a central lumen (
There are a few case studies on the passage of a colonic cast as a result of colonic hypoperfusion. Most patients had ischaemic injury following bowel resection or as a complication of abdominal aortic aneurysms (or aneurysm surgery).