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Arlette Darfeuille-Michaud: Researcher, Lecturer, Leader, Mentor, and Friend

It is with profound sadness that we note the loss, at age 59, of our dear friend and mentor Dr Arlette Darfeuille-Michaud on June 28, 2014, following a 15-month battle against cancer. Arlette was a Professor at Auvergne University, and director of an INSERM unit that investigates microbial-host interactions in intestinal inflammation, located in Clermont-Ferrand, France. Arlette advanced our understanding of intestinal diseases, and humanity in general, via pioneering research in gut microbiology, visionary leadership, energetic teaching, and exceptionally supportive mentorship.

Arlette lived her early years in the tiny French village of Beyssenac (in the beautiful Corrèze region) where her family owned and operated a fruit orchard. Arlette earned her PhD in 1987 at Auvergne University in the lab of Dr Bernard Joly, studying Klebsiella pneumoniae strains involved in nosocomial infections and Escherichia coli pathogenesis. Arlette then joined...


Alcoholic hepatitis (AH) is a severe form of alcoholic liver disease that is associated with high mortality. Despite extensive research on AH over the last several decades, the pathogenesis of AH remains largely unknown, and there are no approved targeted therapies. Inflammation (neutrophil infiltration) is generally believed to play an important role in the pathogenesis of AH; however, the mechanisms underlying neutrophil infiltration in AH and the functions of neutrophils in AH are not fully understood.1 Despite its obscure role, inflammation has been actively investigated as a therapeutic target for the treatment of AH. For example, corticosteroids, which are broadly immunosuppressive drugs, have been widely used for AH therapy for more than five decades. However, the results have been controversial. It is generally accepted that corticosteroid therapy improves short-term, but not long-term, survival rates in patients with severe AH.2

Recently, we have demonstrated that...


Symptoms compatible with IBS, that is, abdominal pain and discomfort, bloating, abdominal distention and an erratic bowel function, are very common reasons for GI consultations in primary care1 and patients with IBS are one of the most frequent patient categories in gastroenterology outpatient clinics.2 Despite being very common and in spite of considerable research effort during the last decades, the pathophysiology of IBS is still considered to be complex and incompletely understood3 4 and even though our understanding of this disabling condition arguably has increased tremendously, we still cannot use knowledge from pathophysiology studies to subgroup IBS patients in a clinically meaningful way. In everyday clinical work, we still rely on subgrouping patients based on the predominant bowel habit when we decide how to manage and treat the patients.5 Even though we have new pharmacological treatment options targeting specific...


Until recently, training of endoscopists was largely up to the local environment, often unstructured and only sporadically supervised. Consequently, there were large variations in the performance of endoscopy, and the quality of the service was very often too poor.1 During recent years, some countries have made great efforts to formalise and structure endoscopist training, often within the framework of continual quality improvement initiatives. This has led to impressive and sustained improvement in the quality of endoscopy services, as recently shown for colonoscopies in the UK.2

The number of procedures performed during training or practice has long been used as a measure of competency. Clearly, competency of technical skills is related to how often someone does something. Repetition (increasing the number of times one does something within a certain amount of time) is a well recognised tool of learning and training, from simple tasks in elementary...


Hepatocellular carcinoma (HCC) is a global health concern. The sixth most common neoplasm, HCC yields a poor prognosis and high mortality rate, causing approximately 500 000 deaths per year. Chronic viral hepatitis—especially HBV and HCV—are major aetiologies of HCC, accounting for approximately 80% of HCC cases, particularly in endemic areas. Another clinical feature of HCC is that it disproportionately occurs in men, and the ratio of male to female patients ranges from 2 to 7:1.1 In large cohort studies, this predominance is only partly explained by gendered differences in environmental influences and behaviours, for example, carcinogen exposure and alcohol consumption; this implies that host genetic factors are crucial determinants. One Taiwanese study revealed that higher serum androgen levels and more active androgen receptor (AR) alleles were associated with an increased risk of HCC among male carriers of HBV, but not men infected with HCV.2 The...


Myeloid-derived suppressor cells (MDSC) represent an immune cell subset with profound immune suppressor function. Initially described in 1978 as natural suppressor cells, this cell type has gained significant attention in recent years not only among tumour immunologists but also among medical oncologists interested both in developing new immune-based antitumour strategies and trying to understand how conventional non-immunological therapies affect antitumour immune responses.1 MDSC represent a mixture of immature cell types that accumulate in tumour-bearing mice and patients with cancer. Tumour-derived factors block the maturation of myeloid cells at different stages, leading to accumulation of these cells in tumours, as well as in the periphery. At least two different subsets of MDSC have been described in mice and men: monocytic and polymorphonuclear granulocytic MDSC, which can be identified as CD14+HLA-DRlo/neg and linnegCD11b+HLA-DRneg in patients2 and as CD11b+Ly6G+ Ly6Clow or CD11b+Ly6Gneg Ly6Chi in mice.3

An...


Aspirin is the most widely studied drug for chemoprevention of colorectal cancer but there are limitations. First the risk-benefit issue has not been fully addressed. What is the risk of bleeding associated with prescribing aspirin to asymptomatic individuals versus the benefit of cancer protection one can obtain from this treatment? Second, the optimal dosing, frequency and duration of aspirin usage in average-risk subjects (ie, patients with non-cardiovascular disease and patients with non-familial polyposis) have not been settled. Third, the potential ethnic difference has not been studied. Up to now, almost all studies were conducted in the Western population. Would Asians be responding to the treatment equally well?

There are new and important data to support the use of aspirin in preventing colorectal cancer (CRC). Rothwell et al recently published their long-term data in 20-year follow-up studies giving strong support to the use of low-dose aspirin (75–300 mg daily) in cancer...


In recent years, an increasing number of reports have shown the involvement of osteopontin (OPN), a pleiotropic cytokine and an important component of the extracellular matrix (ECM), in the pathogenesis of liver injury and the development of fibrosis.1 2 OPN is also frequently overexpressed in hepatocellular carcinoma (HCC), where it modulates HCC growth, invasion and metastasis,2 and in cholangiocarcinoma, where its expression bears prognostic significance. Previous studies3 have shown that in injured livers OPN is expressed by hepatic stellate cells (HSC) and upregulates collagen I production. Interestingly, in HSC, OPN expression appears to be downstream of SOX9, a Hedgehog- and Notch-controlled transcription factor that is expressed also in biliary cells and hepatic progenitor cells (HPC)/hepatocytes committed to the biliary fate.4–6 In this issue of Gut, two papers investigate the role of OPN in HPC-driven ductular...


Thiopurines have been considered the reference treatment for patients with steroid-dependent moderate to severe IBD for many years. This has been based on evidence describing the efficacy and safety of azathioprine and mercaptopurine in Crohn's disease (CD) and UC. In CD, efficacy has been established through controlled trials, meta-analyses, cohort studies and data on mucosal healing, as well as in withdrawal studies. A Cochrane meta-analysis concluded that azathioprine and mercaptopurine effectively maintained remission in CD with an OR between 2 and 3 compared to placebo.1 Mucosal healing was initially studied in patients on azathioprine with longstanding steroid-free remission.2 In this setting, full mucosal healing was achieved in 54% of patients with ileitis and 70% with colitis. The majority of the remaining patients had at least partial healing, and only a small minority experienced no healing at all. This healing potential was confirmed in a...


Background

Oesophageal cancer is one of the most deadly forms of cancer worldwide. Long non-coding RNAs (lncRNAs) are often found to have important regulatory roles.

Objective

To assess the lncRNA expression profile of oesophageal squamous cell carcinoma (OSCC) and identify prognosis-related lncRNAs.

Method

LncRNA expression profiles were studied by microarray in paired tumour and normal tissues from 119 patients with OSCC and validated by qRT-PCR. The 119 patients were divided randomly into training (n=60) and test (n=59) groups. A prognostic signature was developed from the training group using a random Forest supervised classification algorithm and a nearest shrunken centroid algorithm, then validated in a test group and further, in an independent cohort (n=60). The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis.

Results

LncRNAs showed significantly altered expression in OSCC tissues. From the training group, we identified a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENST00000547963.1) which classified the patients into two groups with significantly different overall survival (median survival 19.2 months vs >60 months, p<0.0001). The signature was applied to the test group (median survival 21.5 months vs >60 months, p=0.0030) and independent cohort (median survival 25.8 months vs >48 months, p=0.0187) and showed similar prognostic values in both. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with OSCC. Stratified analysis suggested that the signature was prognostic within clinical stages.

Conclusions

Our results suggest that the three-lncRNA signature is a new biomarker for the prognosis of patients with OSCC, enabling more accurate prediction of survival.


Objective

The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach.

Design

We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors.

Results

The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum.

Conclusions

Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations.


Background

Serum infliximab trough levels correlate with efficacy; dose escalation is often beneficial in patients with Crohn's disease who stop responding to infliximab treatment.

Objective

To carry out a post hoc analysis of A Crohn's Disease Clinical Trial Evaluating Infliximab in a New Long-term Treatment Regimen I (ACCENT I) to evaluate the association between serum infliximab trough levels and C-reactive protein (CRP) after 14 weeks of induction treatment with durable sustained long-term response (Crohn's Disease Activity Index decrease ≥70 points and reduction ≥25% from baseline).

Design

ACCENT I was a multicentre, randomised, placebo-controlled study. Week 14 trough levels and CRP percentage decrease from baseline to week 14 were compared between patients with and without durable sustained response through week 54. Sensitivity and specificity were determined to predict durable sustained response. Receiver operating characteristic (ROC) curves identified optimal cut-off points; logistic regression determined ORs.

Results

After induction with 5 mg/kg infliximab, 25% (37/147) and 33% (47/144) of patients sustained week 14 response to infliximab 5 or 10 mg/kg, respectively, administered every 8 weeks without dose escalation, through week 54. Median week 14 trough levels of patients with and without durable sustained response to infliximab 5 mg/kg were 4.0 and 1.9 μg/mL, respectively (p=0.0331). Optimal predictors of durable sustained response to maintenance infliximab 5 mg/kg were week 14 trough level ≥3.5 µg/mL and ≥60% CRP decrease (ORs (95% CI), 3.5 (1.1 to 11.4) and 7.3 (1.4 to 36.7)), respectively, in patients with raised baseline CRP (>8.0 mg/L); area under the ROC curve was 0.75 for both predictors. A ≥3.5 µg/mL week 14 infliximab serum level did not predict durable sustained response to 10 mg/kg maintenance infliximab.

Conclusions

Patients with durable sustained response to maintenance infliximab 5 mg/kg had higher postinduction trough levels than patients without durable sustained response. Serum infliximab trough levels ≥3.5 µg/mL and ≥60% CRP decrease were significantly associated with durable sustained response.


Objective

Previous studies have shown that ulcerative colitis (UC) is associated with the presence of lamina propria non-invariant (Type II) NKT cells producing IL-13 and mediating epithelial cell cytotoxicity. Here we sought to define the antigen(s) stimulating the NKT cells and to quantitate these cells in the UC lamina propria.

Design

Detection of Type II NKT cells in UC lamina propria mononuclear cells (LPMC) with lyso-sulfatide loaded tetramer and quantum dot-based flow cytometry and staining. Culture of UC LPMCs with lyso-sulfatide glycolipid to determine sulfatide induction of epithelial cell cytotoxicity, IL-13 production and IL-13Rα2 expression. Blinded quantum dot-based phenotypic analysis to assess UC LPMC expression of IL-13Rα2, CD161 and IL-13.

Results

Approximately 36% of UC LPMC were lyso-sulfatide tetramer positive, whereas few, if any, control LPMCs were positive. When tested, the positive cells were also CD3 and IL-13Rα2 positive. Culture of UC LPMC with lyso-sulfatide glycolipid showed that sulfatide stimulates UC LPMC production of IL-13 and induces UC CD161 LPMC-mediated cytotoxicity of activated epithelial cells; additionally, lyso-sulfatide induces enhanced expression of IL-13Rα2. Finally, blinded phenotypic analysis of UC LP MC using multicolour quantum dot-staining technology showed that approximately 60% of the LPMC bear both IL-13Rα2 and CD161 and most of these cells also produce IL-13.

Conclusions

These studies show that UC lamina propria is replete with Type II NKT cells responsive to lyso-sulfatide glycolipid and bearing IL-13Rα2. Since lyso-sulfatide is a self-antigen, these data suggest that an autoimmune response is involved in UC pathogenesis.


A 27-year-old Caucasian male presented with a one-month history of diarrhoea, severe abdominal cramping, heartburn and weight loss. The symptoms began right after breakfast in the morning and continued with 3–4 watery stools throughout the day, but none during the night. Spicy foods, alcoholic beverages and excessive water intake seemed to exacerbate the pain, but soda seemed to alleviate it. The patient denied use of antibiotics, antispasmodics, or antihypertensive drugs, and drank minimal milk. The patient denied any recent international travel or contact with diapers or ill persons but confirmed that the symptoms started about a week after a river trip. There was no known family history of GI disease. Endoscopically, the oesophageal, gastric, small bowel and colonic mucosa appeared normal. Multiple cold forceps biopsies were performed for histology in the lower third of the oesophagus, second part of the duodenum, terminal ileum (TI) and random colon....


Background

About 10% of patients with IBS report the start of the syndrome after infectious enteritis. The clinical features of postinfectious IBS (PI-IBS) resemble those of diarrhoea-predominant IBS (IBS-D). While altered faecal microbiota has been identified in other IBS subtypes, composition of the microbiota in patients with PI-IBS remains uncharacterised.

Objective

To characterise the microbial composition of patients with PI-IBS, and to examine the associations between the faecal microbiota and a patient's clinical features.

Design

Using a phylogenetic microarray and selected qPCR assays, we analysed differences in the faecal microbiota of 57 subjects from five study groups: patients with diagnosed PI-IBS, patients who 6 months after gastroenteritis had either persisting bowel dysfunction or no IBS symptoms, benchmarked against patients with IBS-D and healthy controls. In addition, the associations between the faecal microbiota and health were investigated by correlating the microbial profiles to immunological markers, quality of life indicators and host gene expression in rectal biopsies.

Results

Microbiota analysis revealed a bacterial profile of 27 genus-like groups, providing an Index of Microbial Dysbiosis (IMD), which significantly separated patient groups and controls. Within this profile, several members of Bacteroidetes phylum were increased 12-fold in patients, while healthy controls had 35-fold more uncultured Clostridia. We showed correlations between the IMD and expression of several host gene pathways, including amino acid synthesis, cell junction integrity and inflammatory response, suggesting an impaired epithelial barrier function in IBS.

Conclusions

The faecal microbiota of patients with PI-IBS differs from that of healthy controls and resembles that of patients with IBS-D, suggesting a common pathophysiology. Moreover, our analysis suggests a variety of host–microbe associations that may underlie intestinal symptoms, initiated by gastroenteritis.


Objective

The number of colonoscopies required to reach competency is not well established. The primary aim of this study was to determine the number of colonoscopies trainees need to perform to attain competency, defined by a caecal intubation rate (CIR) ≥90%. As competency depends on completion, we also investigated trainee factors that were associated with colonoscopy completion.

Design

The Joint Advisory Group on GI Endoscopy in the UK has developed a trainee e-portfolio from which colonoscopy data were retrieved. Inclusion criteria were all trainees who had performed a total of ≥20 colonoscopies and had performed ≤50 colonoscopies prior to submission of data to the e-portfolio. The primary outcome measure was colonoscopy completion. The number of colonoscopies required to achieve CIR ≥90% was calculated by the moving average method and learning curve cumulative summation (LC-Cusum) analysis. To determine factors which determine colonoscopy completion, a mixed effect logistic regression model was developed which allowed for nesting of patients within trainees and nesting of patients within hospitals, with various patient, trainee and training factors entered as fixed effects.

Results

297 trainees undertook 36 730 colonoscopies. By moving average analysis, the cohort of trainees reached a CIR of 90% at 233 procedures. By LC-Cusum analysis, 41% of trainees were competent after 200 procedures. Of the trainee factors, the number of colonoscopies, intensity of training and previous flexible sigmoidoscopy experience were significant factors associated with colonoscopy completion.

Conclusions

This is the largest study to date investigating the number of procedures required to achieve competency in colonoscopy. The current training certification benchmark in the UK of 200 procedures does not appear to be an inappropriate minimum requirement. The LC-Cusum chart provides real time feedback on individual learning curves for trainees. The association of training intensity and flexible sigmoidoscopy experience with colonoscopy completion could be exploited in training programmes.


Objective

To evaluate the influence of low-dose, enteric-coated aspirin tablets (100 mg/day for 2 years) on colorectal tumour recurrence in Asian patients with single/multiple colorectal tumours excised by endoscopy.

Design

A double-blinded, randomised, placebo-controlled multicentre clinical trial was conducted.

Participants

311 subjects with single/multiple colorectal adenomas and adenocarcinomas excised by endoscopy were enrolled in the study (152 patients in the aspirin group and 159 patients in the placebo group). Enrolment began at the hospitals (n=19) in 2007 and was completed in 2009.

Results

The subjects treated with aspirin displayed reduced colorectal tumourigenesis and primary endpoints with an adjusted OR of 0.60 (95% CI 0.36 to 0.98) compared with the subjects in the placebo group. Subgroup analysis revealed that subjects who were non-smokers, defined as those who had smoked in the past or who had never smoked, had a marked reduction in the number of recurrent tumours in the aspirin-treated group. The adjusted OR for aspirin treatment in non-smokers was 0.37 (CI 0.21 to 0.68, p<0.05). Interestingly, the use of aspirin in smokers resulted in an increased risk, with an OR of 3.44. In addition, no severe adverse effects were observed in either group.

Conclusions

Low-dose, enteric-coated aspirin tablets reduced colorectal tumour recurrence in an Asian population. The results are consistent with those obtained from other randomised controlled trials in Western countries.

The clinical trial registry website and the clinical trial number

http://www.umin.ac.jp (number UMIN000000697).


Background

Oral administration of the omega-3 fatty acid eicosapentaenoic acid (EPA), as the free fatty acid (FFA), leads to EPA incorporation into, and reduced growth of, experimental colorectal cancer liver metastases (CRCLM).

Design:

We performed a Phase II double-blind, randomised, placebo-controlled trial of EPA-FFA 2 g daily in patients undergoing liver resection surgery for CRCLM. The patients took EPA-FFA (n=43) or placebo (n=45) prior to surgery. The primary end-point was the CRCLM Ki67 proliferation index (PI). Secondary end-points included safety and tolerability of EPA-FFA, tumour fatty acid content and CD31-positive vascularity. We also analysed overall survival (OS) and disease-free survival (DFS).

Results

The median (range) duration of EPA-FFA treatment was 30 (12–65) days. Treatment groups were well matched with no significant difference in disease burden at surgery or preoperative chemotherapy. EPA-FFA treatment was well tolerated with no excess of postoperative complications. Tumour tissue from EPA-FFA-treated patients demonstrated a 40% increase in EPA content (p=0.0008), no difference in Ki67 PI, but reduced vascularity in ‘EPA-naïve’ individuals (p=0.075). EPA-FFA also demonstrated antiangiogenic activity in vitro. In the first 18 months after CRCLM resection, EPA-FFA-treated individuals obtained OS benefit compared with placebo, although early CRC recurrence rates were similar.

Conclusions

EPA-FFA therapy is safe and well tolerated in patients with advanced CRC undergoing liver surgery. EPA-FFA may have antiangiogenic properties. Remarkably, limited preoperative treatment may provide postoperative OS benefit. Phase III clinical evaluation of prolonged EPA-FFA treatment in CRCLM patients is warranted.

Trial Identifier:

ClinicalTrials.gov NCT01070355.


Background

Pancreatic ductal adenocarcinoma (PDA) is characterised by a robust desmoplasia, including the notable accumulation of immunosuppressive cells that shield neoplastic cells from immune detection. Immune evasion may be further enhanced if the malignant cells fail to express high levels of antigens that are sufficiently immunogenic to engender an effector T cell response.

Objective

To investigate the predominant subsets of immunosuppressive cancer-conditioned myeloid cells that chronicle and shape the progression of pancreas cancer. We show that selective depletion of one subset of myeloid-derived suppressor cells (MDSC) in an autochthonous, genetically engineered mouse model (GEMM) of PDA unmasks the ability of the adaptive immune response to engage and target tumour epithelial cells.

Methods

A combination of in vivo and in vitro studies were performed employing a GEMM that faithfully recapitulates the cardinal features of human PDA. The predominant cancer-conditioned myeloid cell subpopulation was specifically targeted in vivo and the biological outcomes determined.

Results

PDA orchestrates the induction of distinct subsets of cancer-associated myeloid cells through the production of factors known to influence myelopoiesis. These immature myeloid cells inhibit the proliferation and induce apoptosis of activated T cells. Targeted depletion of granulocytic MDSC (Gr-MDSC) in autochthonous PDA increases the intratumoral accumulation of activated CD8 T cells and apoptosis of tumour epithelial cells and also remodels the tumour stroma.

Conclusions

Neoplastic ductal cells of the pancreas induce distinct myeloid cell subsets that promote tumour cell survival and accumulation. Targeted depletion of a single myeloid subset, the Gr-MDSC, can unmask an endogenous T cell response, disclosing an unexpected latent immunity and invoking targeting of Gr-MDSC as a potential strategy to exploit for treating this highly lethal disease.


Objective

Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury.

Design

CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo.

Results

CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs.

Conclusions

Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.


Background

Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure.

Objective

To determine the molecular function of CCRK in HBV-associated HCC.

Design

Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel–Cox test.

Results

Overexpression of CCRK, but not its kinase-defective mutant, activated β-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3β/β-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3β phosphorylation at Ser9, active dephosphorylated β-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates.

Conclusions

Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.


Objective

In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring.

Results

In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-β+ BEC, which were lacking in TAA-treated Opn–/– mice and in healthy human explants, this suggested that OPN could regulate TGF-β, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-β expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-β (rTGFβ) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-β reduced collagen-I expression in co-cultured HSC.

Conclusions

OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-β.


Basic scienceA novel method of liver preservation may alleviate the shortage of donor organs

 Berendsen TA, Bruinsma BG, Puts CF, et al. Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nat Med 2014;20:790–3.

There is a shortage of donor organs for transplantation worldwide. One strategy to increase availability is to enhance organ preservation and allow more potentially available organs to be used successfully. Currently, viable preservation is available up to 12 h. Although various methods of supercooling to enhance preservation have been assessed, there are many challenges in applying this to whole organs and thus far this approach has been relatively unsuccessful in animal models. This current paper reports a novel 4-step method to extend preservation of fresh rat liver to beyond 72 h and still achieve successful transplantation outcome. The method employs supercooling, the use of 2 cryoprotectants (polyethylene glycol for extracellular cell membrane protection and...


The research on colorectal cancer (CRC) biology has been leading the oncology field since the early 1990s. The search for genetic alterations has allowed the identification of the main tumour suppressors or oncogenes. Recent work obtained in CRC has unexpectedly proposed the existence of novel category of tumour suppressors, the so-called ‘dependence receptors’. These transmembrane receptors behave as Dr Jekyll and Mr Hyde with two opposite sides: they induce a positive signalling (survival, proliferation, differentiation) in presence of their ligand, but are not inactive in the absence of their ligand and rather trigger apoptosis when unbound. This trait confers them a conditional tumour suppressor activity: they eliminate cells that grow abnormally in an environment offering a limited quantity of ligand. This review will describe how receptors such as deleted in colorectal carcinoma (DCC), uncoordinated 5 (UNC5), rearranged during transfection (RET) or TrkC constrain CRC progression and how this dependence receptor paradigm may open up therapeutical perspectives.


Sir,

We read with interest the article by Uhlig on the Mendelian forms of IBD.1 Mendelian forms of Crohn's disease (CD) are rare but can establish a causal link that is not usually possible by the genome-wide association study (GWAS) design. In order to accelerate the discovery of Mendelian CD, we recruited four cooperative families in which the healthy consanguineous parents had at least two affected children with early onset CD (<10 years of age), consistent with autosomal recessive inheritance (see online supplementary figure S1). With informed consent according to an Institutional Review Board approved protocol (KFSHRC RAC# 2121053), we proceeded with autozygosity mapping using AgileMultiIdeogram (http://dna.leeds.ac.uk/agile/AgileMultiIdeogram/) followed by whole-exome sequencing (WES) as previously described.2 Online supplementary table S1 shows the iterative filtering of the resulting variants based on homozygosity, predicted pathogenicity, location within the autozygome and novelty.

Family 1 consists of...