Christine Weber

Neurogastroenterology: Improving glucose tolerance via the gut-brain axis.

neurons via bioactive peptides present in the gut lumen. In addition, we discovered a new mode of communication between the gut and the brain (mechanical contraction) to control glycaemia,” summarize Knauf and Cani. “We aim to find potential novel molecular actors (peptides, nutrients or others) present in the lumen able to modulate ENS activity. We hope that our findings will contribute to propose new therapeutic strategies to treat hyperglycaemia associated with T2DM, preferentially via an oral route.” Christine Weber Type 2 diabetes mellitus (T2DM) is associated with insulin resistance, altered intestinal glucose sensing and hypercontractility. In this study, Fournel and colleagues now present a mechanism by which apelin, a bioactive peptide known to aid glucose absorption, ameliorates glucose tolerance by targeting the enteric nervous system (ENS) and decreasing duodenal contraction in mice. “Previous work from our team demonstrates that glucose sensing in the intestine modulates the gut–brain axis, and this is associated with modulation of glucose entry in skeletal muscle. However, the mechanisms are poorly understood,” explain corresponding authors Claude Knauf and Patrice Cani. By measuring mechanical contractions ex vivo in duodenal preparations and electrical duodenal activity in vivo in mice, Fournel et al. confirmed that apelin is indeed able to control duodenal motility. In the intestine, apelin dose-dependently modified acetylcholine and nitric oxide (NO) release by ENS neurons. NO is a central factor involved in glucose metabolism. Whereas 100 nM of oral apelin increased duodenal contractility and decreased glucose entry into the muscle (but not into the liver or adipose tissue) via the hypothalamic NO relay, chronic oral treatment with 1 μM of apelin for a week exerted a contrasting effect. High dose apelin decreased basal duodenal contractility, led to a more pronounced insulin release in response to glucose, increased oral glucose tolerance and an upregulation of glucose transporter type 4 mRNA expression and glucose entry into the muscle. In mice fed a high-fat diet, the resulting hypercontractility of the duodenum could be counterbalanced by treatment with 1 μM of apelin, which induced increased NO release and improved glucose utilization. Chronic treatment for 1 week improved glycaemia, insulin resistance, glucose tolerance and glucose entry into the muscle. “Our work proposes a novel approach: targeting the ENS N E U R O G A S T R O E N T E R O LO GY

Biomarkers: The challenge to find biomarkers for the early detection of pancreatic cancer

The challenge to find biomarkers for the early detection of pancreatic cancer Promising advances are currently being made in the search for reliable biomarkers that can facilitate the early detection of pancreatic cancer. In a study published in Nature, evidence was presented that glypican-1 (GPC1) expression on pancreatic-cancer-derived exosomes can be used to detect early pancreatic cancer and gain insights into disease progress and tumour burden. Exosomes are formed when intracellular multivesicular endosomes bulge inwards to create small, membranous vesicles. These endosomes fuse with the plasma membrane, releasing exosomes into the extracellular environment and circulation where they can be detected using blood tests. Exosomes contain proteins and nucleic acids, and their composition varies widely depending on the type and condition of the cell they originate from, making them an ideal, easily accessible reservoir of cancer biomarkers to assess disease processes. “We had an interest in understanding the biology of exosomes and their functions in cancer,” explains corresponding author Raghu Kalluri. His research group analysed the protein contents of exosomes originating from pancreatic cancer cell lines and control cells using chromatography-mass spectrometry and flow cytometry. When they compared pancreatic cancer exosomes with those from non-tumorigenic cell lines, the cancer exosomes showed elevated levels of GPC1, a protein that has previously been reported as overexpressed in these cancers. In blood samples from human patients with pancreatic ductal adenocarcinoma (PDAC, n = 190), substantially higher levels of circulating GPC1+ exosomes were detected than in healthy donors (n = 100). In addition, the investigators assessed the tumour material for oncogenic KRAS mutations, a common feature in PDAC. In the same patients (n = 15) who harboured these variants in their tumour genome, GPC1+ exosomes also contained identical KRAS mutations. However, the presence of oncogenic DNA in cancer exosomes was only evaluated in a subset of the whole patient population. Interestingly, levels of circulating GPC1+ exosomes were higher in patients with histologically confirmed pancreatic cancer precursor lesions than in patients with benign pancreatic disease or healthy individuals. The efficiency of GPC1+ exosomes in distinguishing benign from neoplastic lesions and healthy individuals exceeded that of carbohydrate antigen 19-9 (CA19-9), a widely used tumour marker in PDAC. Tumour burden could also be assessed: higher levels of GPC1+ exosomes were found in patients with PDAC and distant metastatic disease than in patients with metastases in lymph nodes or no metastases. In most patients exosome levels decreased after surgical resection of the tumour. A reduction in circulating GPC1+ exosome levels was also correlated with increased survival in these patients. When tested in a genetically engineered mouse model of PDAC that specifically recapitulates features of human disease progression, the investigators could show that Gpc1+ exosome levels increase proportionally with time and tumour burden, as the disease progresses from early neoplastic stages to malignant disease. Reassuringly, an increase in Gpc1+ exosome levels is evident before the mice develop pancreatic masses. By contrast, healthy mice retain a low level of Gpc1+ exosomes during all time points, similarly to a mouse model of acute pancreatitis, which argues for cancer specificity of the test. Researchers estimate that pancreatic cancer requires 15–20 years to evolve from early lesions to PDAC; even though lesions have to progress to certain stages to become detectable, a sufficient time window for cancer prevention might be achievable if suitable biomarkers are found. Currently, ~80% of patients are diagnosed when PDAC has already progressed to metastatic disease. “Patients with very early stage disease are most likely to benefit from an early detection test,” says Michael Goggins (Johns Hopkins University), who was not involved in the research. He acknowledges the study’s potential strengths—high sensitivity of the test, the methods used to identify exosomes, and large study populations with resectable pancreatic cancer—but also the limitations. “Specifically, the authors did not evaluate the test’s performance in a sufficiently sized group of patients with very early stage disease (stage 1 pancreatic cancer or carcinoma-in-situ).” However, these limitations also reflect the dilemma the researchers had set out to solve. “It is not surprising they had few patients with stage 1 pancreatic cancer because it is very difficult to identify patients with stage 1 cancers,” says Goggins. Although the findings from experiments in mouse models look promising, more meticulous work will be needed to confirm the efficiency of biomarkers in the early detection of cancerous lesions in humans. “We hope to test many more patients to validate our findings and to diagnose and track patients with pancreatic cancer,” concludes Kalluri.

Oesophageal cancer: Neoadjuvant chemoradiotherapy and surgery superior to surgery alone

Long-term results of a randomized controlled trial on effects of surgery plus chemoradiotherapy versus surgery alone in patients with oesophageal cancer have confirmed the initially reported survival benefit of combined therapy. Patients (n = 368) with clinically resectable, locally advanced cancer of the oesophagus or oesophagogastric junction were enrolled to receive either neoadjuvant chemoradiotherapy (intravenous carboplatin or paclitaxel) with concurrent radiotherapy followed by surgery, or surgery alone. Surviving patients were followed up for a median of 84.1 months. In the group of patients who were treated with chemoradiotherapy and surgery, the median overall survival was 48.6 months (95% CI 32.1–65.1), whereas patients who only received surgery had a median overall survival of 24.0 months (95% CI 14.2–33.7; P = 0.003). Differences between cancer subtypes were also reported. Median OESOPHAGEAL CANCER

Liver: Neutrophil extracellular traps mediate bacterial liver damage

A new study in Nature Communications reports on the contribution of neutrophil extracellular traps (NETs) to liver damage after methicillin-resistant Staphylococcus aureus (MRSA) infection. Neutrophils are part of the first line of defence during bacterial infection and they have a number of strategies at their disposal to efficiently deal with pathogens. Phagocytosis and degranulation are the mechanisms they are probably best known to use, but they also employ NETs to—quite literally—trap bacteria. NETs comprise DNA webs with attached histones and granule enzymes, such as neutrophil elastase (NE). In addition, peptidyl arginine deiminase type IV (PAD4) also has an essential role in NET formation. Kolaczkowska et al. showed that, after MRSA infection, bacteria accumulated in the liver more readily than in other organs, as they were captured by Kupffer cells residing in the liver. Subsequently recruited neutrophils released NETs into the vasculature in an attempt to eradicate the pathogens; a response that might actually do more harm than good. “We found that the majority of the injury in the liver was induced by these NETs and not by the bacteria itself,” says corresponding author Paul Kubes. Liver damage was attenuated when MRSA-mediated NET release was reduced in mice via elastase inhibition or genetic ablation of Ne or Pad4. “We also found that NETs could linger around for quite some time, attached to vessel walls,” explains Kubes. The authors identified von Willebrand factor as an anchor that equally helped to reduce liver damage when disrupted. However, deoxyribonuclease (DNAse) enzymes, which can dissolve NETs by breaking up their DNA backbone, might not be applicable to prevent liver damage. DNAse fails to clear all NET components, leaving histones and NE attached to the endothelium. Despite DNAse treatment, NE retains its proteolytic activity and damages the surrounding tissue. The authors caution that this effect could have clinical implications as DNAse is used therapeutically. They propose that inhibition of NET formation or shedding of anchor molecules might prove a more effective strategy in damage-prevention than NET dissolution.

Nutrition: Diet change alters microbiota and might affect cancer risk

differences in the gut microbiota of African Americans and rural Africans are believed to contribute to the disparity in cancer incidence. The authors wanted to clarify whether a diet change could potentially reverse these microbial profiles and perhaps even influence cancer risk. In their study, they switched rural Africans from their normal diet to a ‘westernized’ high-fat, low-fibre food regime and African Americans to a lowfat, high-fibre ‘African diet’ for 2 weeks. All participants underwent colonoscopy, and had faecal and colonic mucosal samples taken before and after diet change. Mucosal samples were analysed for proliferation and inflammation markers, two indicators of a potentially elevated cancer risk. Although short in duration, the food interventions showed remarkable effects. Compared with their normal diet, African Americans demonstrated reduced proliferation and inflammation when switched to the ‘African diet’. Conversely, rural Africans on a ‘westernized diet’ exhibited increased NUTRITION

Gut microbiota: Commensal bacteria and intestinal surgery complications

community and identified two species, including E. faecalis that demonstrated collagenase and MMP9-activating properties. These bacteria were not killed by the standard intravenous antibiotics given to the patients before surgery. “Now with this evidence in hand, we can do something about [anastomotic leaks]; especially since our work shows that the antibiotics we use and the way we use them is probably inadequate,” emphasizes Alverdy.

Liver: Reprogramming a hepatocyte's memory of liver disease

A new study published in The Journal of Clinical Investigation demonstrated the downregulation of a network of hepatocyteenriched transcription factors in damaged hepatocytes that had been isolated from a rat model of chronic liver injury. Conversely, the forced re-expression of one factor, hepatocyte nuclear factor 4 alpha (Hnf4a), improved cellular function and reversed liver failure. Carbon tetrachloride (CCl4) administration in rats reproduces aspects of advanced degenerative liver disease seen in humans, including irreversibly decompensated hepatocytes. Using this model, Nishikawa and colleagues found that in association with endstage chronic liver disease, a network of transcription factors expressed within hepatocytes were downregulated; these factors included those important to hepatocyte development such as Hnf4a, Foxa2, Cebpa, Ppara and Hnf1a. The effect could still be observed 4 weeks after CCl4 discontinuation. “We wondered whether the damaged cells could be reprogrammed in the same way scientists have been able to reprogramme somatic cells into stem cells or into cells representing other lineages,” explains corresponding author Ira Fox (University of Pittsburgh, PA, USA). Re-expression of Hnf4a in cultured hepatocytes from animals with fatally decompensated liver function restored the expression of several other previously downregulated transcription factors and improved cell function. In the rat model of liver failure, hepatocytes were ‘reprogrammed’ by injecting animals with adeno-associated viral vectors expressing Hnf4a. This treatment prolonged the survival of injected animals and improved liver function compared with untreated rats, as measured by an increase in clotting factors and liver protein expression. When isolated, hepatocytes from treated animals exhibited normalized cell function and gene expression compared with cells from control animals. Interestingly, reprogrammed hepatocytes did not demonstrate increased proliferation, although reduced apoptotic cell death was evident. “Our studies showed that the animals didn’t get better as a result of stem cell activation or regeneration by growing new liver cells. Instead, the diseased cells had healed,” says Fox. The precise molecular mechanism responsible for the disruption of the hepatic transcription factor network in liver injury remains to be determined. Likewise, it is not clear why dysfunctional hepatocytes cannot seem to restore normal gene function independently. “We are now looking for the signal pathways that cause hepatocytes to fail and other pathways that allow restored hepatocytes to resolve fibrosis,” concludes Fox. The authors hope that these investigations will provide therapeutic candidates for advanced drugs that can specifically target pathways mediating liver fibrosis and hepatocyte damage.

Intestinal tract: DLL4 signalling maintains organization and function of lacteals

maintenance and regeneration of lacteals, specialized lymphatic vessels in the villi of the small intestine, have been reported in a recent study published in The Journal of Clinical Investigation. Although often overlooked in research, the intestinal lymphatic vasculature adopts key functions in the small intestine, regulates absorption and transport of fatty acids, and encourages an active crosstalk between immune cells in the area. “Understanding mechanisms controlling these specialized blood and lymphatic vessels in adult organs where pathologies arise is critical for novel therapies,” explain first author Jeremiah BernierLatmani and corresponding author Tatiana Petrova. Using a whole-mount immunostaining approach, the researchers analysed the stroma of small intestinal villi in mice and revealed a well-structured organization (see image) with blood capillaries that form cage-like patterns (red), interspersed with tree-like smooth muscle fibres (cyan) and lacteals (green) located within the centre. Immune cells, such as regulatory T cells and dendritic cells, were also found in close proximity of the smooth muscle cell fibres. Proliferation rates of lacteal lymphatic endothelial cells (LECs) in adult mice were low but steady (2.5% of Ki67+ cells), whereas in embryonic skin ~30% of LECs were Ki67+. Submucosal LECs were quiescent. “We found that, surprisingly, adult lacteals are constantly and slowly regenerating, because they continuously encounter high mechanical stress, microbial products and dietary fat. Interestingly, such regeneration of lacteals parallels constant regeneration of intestinal epithelium,” the authors note. In an attempt to decipher the molecular mechanisms involved in lacteal homoeostasis and regeneration, the investigators focussed on Notch signalling, which is known to regulate many aspects of endothelial physiology. They detected different levels of the Notch ligand DLL4 ( delta-like protein 4) in arterioles (high), blood capillaries (intermediate) and venules (low) of intestinal villi. Tip cells of sprouting lacteals exhibited high DLL4 levels, indicating a potential role of DLL4 in lacteal function. Deletion of Dll4 in lymphatic vessels of adult mice resulted in a substantially shorter length of the lacteals, impaired LEC survival and reduced LEC migration. By contrast, inactivation of Dll4 in blood vessels increased the number of filopodia and vessel branching. Using blocking antibodies, VEGFR3 and VEGFR2 signalling was confirmed as essential for Dll4 expression and lacteal maintenance. Loss of Dll4 also disrupted the organization of lacteal adherens junctions, probably resulting in impaired fat uptake. “Overall, our work reveals how lymphangiogenic responses are shaped by tissue specialization and report a novel process for matching a unique need of intestinal lymphatic vessels both for continuous repair, and an efficient uptake of fat,” conclude the authors. Considering the important physiological roles of the intestinal lymphatic system, its dysregulation could also contribute to pathology. Future research will focus on additional molecular players involved in the specialization of intestinal lymphatic vessels and their crosstalk with other intestinal cells. Christine Weber I N T E S T I N A L T R AC T

Liver: Macrophage-stimulating CSF1 is important in liver injury

hepatectomy or intoxication with paracetamol. In both these settings, liver tissue is lost acutely in the context of a fairly normal underlying liver, so providing a reasonably controlled environment,” explains corresponding author Stuart Forbes. Results confirm a marked effect: in 55 patients who had up to 75% of their liver tissue removed, CSF1 levels were substantially elevated compared with healthy individuals, and the increase was correlated to the extent of the resection. In patients with paracetamol-induced acute liver failure, increased serum levels of CSF1 were found in those individuals whose livers regenerated, whereas low serum CSF1 levels were associated with patient deterioration. When the investigators compared the value of CSF1 as a predictor of outcome to the current gold-standard biomarker serum acetyl-HMGB1, CSF1 levels demonstrated better correlations. As liver biopsies pose a substantial risk for the patient, some experiments were conducted in mice. Subcutaneous LIVER

Pancreatitis: Alternatively activated macrophages mediate fibrosis.

activated, macrophages produce a range of cytokines resulting in different functions and specializations. To induce chronic pancreatitis, mice were treated with caerulein for 4 weeks. Higher levels of profibrotic and monocyte or macrophage-specific cytokines were evident in mice with chronic pancreatitis than in untreated controls. As expected, macrophages accumulated in the pancreas of treated animals, owing to increased proliferation of resident cells and monocyte recruitment from the bone marrow. The macrophages in mouse and human chronic pancreatitis tissue samples were found to be AAMs, expressing characteristic M2 genes, such as MRC1, IL10, TGFB1 and PDGFB. Isolated PSCs from mice and human patients with chronic pancreatitis secreted cytokines that promoted fibrosis (TGF-β) and AAMs (IL-4, IL-13). These PSCs were also found to actively polarize macrophages into AAMs in co-culture. Alternative activation, in turn, enabled the macrophages to potently activate PSCs, inducing a positive feedback loop that ultimately PANCREATITIS