Can We Treat Diarrhea by Stimulating Na+ Absorption?

Drugs designed to increase intestinal absorption of sodium might be the best approach for treatment of diarrheal diseases, according to the Advances in Translational Science article in the January issue of Clinical Gastroenterology and Hepatology.

Acute diarrheal diseases are the second leading cause of of death in children under 5 years old. Worldwide, there are an estimated 1.7 billion cases, and diarrhea is responsible for killing approximately 760, 000 children every year. In the US,  adults experience 99 million episodes of acute diarrhea or gastroenteritis, resulting in about 8 million physician visits and more than 250,000 hospital admissions each year (1.5% of adult hospitalizations).

There are some drugs for treatment of diarrhea, including opiates, which only moderately decrease stool output; racecadotril, which has inconsistent effects on acute diarrhea in children; and crofelemer—a CI channel inhibitor approved by the FDA for treatment of HIV-associated diarrhea.

However, oral rehydration solutions still account for the greatest reductions in mortality among children in developing countries. These solutions rehydrate patients but don’t necessarily reduce stool output or length of illness. More effective drugs are needed.

Diarrheal diseases develop via altered intestinal transport of electrolytes and water. Epithelial cells in the villus of the small intestine or surface and upper crypt of the colon mostly absorb Na+, whereas those in the lower crypt primarily secrete Cl and HCO3 . Infectious agents that cause diarrhea alter electrolyte transport and intestinal permeability by inhibiting Na+ absorption and stimulating anion and K+ secretion (see figure).

The plasma membrane of each cell is divided into the apical (brush border [BB]) and basolateral (serosal) membrane. Specific membrane transport proteins segregate to either the apical or basolateral side of these cells, and are required for transepithelial electrolyte transport (absorption and secretion). This transport processes contributes to intestinal Na+ absorption. Reagents are needed to either reverse the changes in transport that occur in diarrhea and/or stimulate other transport processes that can compensate for these changes.

The plasma membrane of each intestinal epitheial cell is divided into the apical (brush border) and basolateral membrane. Specific membrane transport proteins segregate to either side of these cells, and are required for electrolyte transport (absorption and secretion), which contributes to intestinal Na+ absorption. Reagents are needed to either reverse the changes in transport that occur in diarrhea and/or stimulate other transport processes to compensate for these changes.

Varsha Singh et al. review drugs that might be used to treat diarrhea by stimulating intestinal Na+ absorption. They describe the molecular mechanisms by which intestinal water and Na+ are absorbed in healthy people and how those processes change during development of diarrhea.

Singh et al. discuss the therapeutic potential of agents designed to stimulate intestinal absorption of Na+. These would alter activities of the brush border Na+/H+ exchanger (NHE3), the Cl–/HCO3– exchanger SLC26A3 (DRA), the Na+ D-glucose linked co-transporter 1 (SGLT1), or the epithelial Na+ channel (ENaC).

Another approach to increase intestinal absorption of Na+ involves addition of zinc to oral rehydration solutions—zinc inhibits stimulated Clsecretion to reduce the duration of diarrhea. Furthermore, the intestinal calcium-sensing receptor (CaSR) regulates intestinal secretion and absorption. Stimulating the CaSR with either calcium or sensitizing (calcimimetic) compounds reverses changes in colonic Na+ absorption and Cl– secretion caused by bacterial toxins.

Singh et al. explain that drugs currently approved to stimulate NaCl absorption are restricted to analogues of physiologic regulators including mu agonist opiates, somatostatin, and clonidine (an alphaadrenergic receptor agonist). These agonists have all been used to treat diarrhea but are limited by side effects, high costs, requirement for parenteral administration, or limited potency. Although modifications of these classes of drugs might be developed, new ways to stimulate Na+ absorption are needed.

However, the authors conclude that drug development is a slow process, and no lead compounds that stimulate neutral NaCl absorption are likely to emerge as useful treatments of diarrhea any time soon.

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Mapping HCV Infection in the Liver

Using single-cell laser capture and high-resolution analysis, researchers show that hepatitis C virus (HCV) infects hepatocytes in the human liver in nonrandom clusters, whereas expression of anti-viral molecules is scattered among hepatocytes. The findings are presented in the December issue of Gastroenterology.

HCV predominantly infects hepatocytes, but most hepatocytes in the liver remain uninfected—HCV antigens have been observed to cluster. This suggests a localized mechanism of intra-hepatic propagation and control. Understanding this process could increase our understanding of infection and strategies for treatment.

Abraham J. Kandathil et al. analyzed liver samples from 4 patients with chronic HCV infection to estimate the proportion of infected hepatocytes and the amount of HCV RNA per cell using single-cell laser capture microdissection (LCM).

LCM unites light microscopy with a low-intensity ultraviolet laser, allowing researchers to ensnare enriched cellular material from tissue samples while preserving positional information, because the tissue is not homogenized. Kandathil et al. made improvements to the technique to increase its resolution, developing single-cell LCM, which allowed them to compare host and viral RNAs.

Studying viral replication in liver tissues from patients with chronic HCV infections, Kandathil et al. estimated the amount of HCV RNA per infected hepatocyte, to determine how the virus spreads in vivo and localize host cell expression of antiviral molecules.

The authors used their data to create a map of viral RNA in hepatocytes, which they called the viroscape. The viroscape shows hepatocytes containing narrow HCV replication peaks surrounded by broad regions with minimal or no HCV vRNA that resembled valleys (see video).

HCV viroscape with superimposed IFITM3 landscape. HCV RNA from 1 patient is represented as contoured gray peaks above the xy plane, and IFITM3 mRNA in the same hepatocytes is represented as contoured terrain below the xy plane.

HCV viroscape with superimposed IFITM3 landscape. HCV RNA from 1 patient is represented as contoured gray peaks above the xy plane, and IFITM3 mRNA in the same hepatocytes is represented as contoured terrain below the xy plane.

Kandathil et al. found that the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of viral RNA ranged from 1 to 50 IU/hepatocyte. However, infection was not random—the authors saw clusters of HCV-positive hepatocytes. These clusters in the hepatic viroscapes indicate cell-to-cell propagation of infection.

Kandathil et al. characterized the spatial association between intrahepatic HCV replication and innate immune signaling, and found that although expression of interferon-stimulated genes was sporadic, it was not specifically targeted toward or away from HCV-positive hepatocytes.

Clustering of HCV-infected hepatocytes did not appear to be caused by short-range immunologic control. IFITM3, an interferon-λ–induced protein that has direct antiviral effects against HCV in cell culture, did not appear to be directed specifically toward or away from infected hepatocytes (green in video).

In liver tissues from some subjects, the author found an association between the peak of viral RNA in a cluster and the number of cells in the cluster. This might suggest that infected hepatocytes depend on the robustness of viral replication in the hepatocyte most permissive to viral replication. Alternatively, the hepatocyte with the highest viral RNA copy number could have been the earliest infected cell of a cluster.

Cell-to-cell propagation of HCV could have important implications for vaccine design and drug development—strategies to inhibit entry of extracellular virions could be insufficient for HCV control if cell-to-cell spread of infection is rampant.

The authors hope for future studies with expanded viroscapes, so they can analyze expression of other host genes that control or support HCV replication.

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Can We Increase Survival of Patients with Gastric Cancer?

Researchers have identified factors that affect life expectancy of patients with stomach cancer, reported in the December issue of Clinical Gastroenterology and Hepatology. Use of these factors to increase early detection of gastric adenocarcinoma could greatly increase survival times among patients.

Gastric cancer is a significant cause of mortality and morbidity worldwide. However, thanks to gastrointestinal endoscopy, cancers can now be detected at early stages, which increases the 5-year survival rate to 90%. Few nationwide long-term assessment studies have estimated the exact potential life-years saved by early detection of gastric cancer, and it’s not clear exactly what factors are associated with survival.

Wei-Ying Chen et al. therefore collected data from 35,576 patients with gastric cancer from the Taiwan Cancer Registry, from 1998 through 2007. They compared patients’ age at diagnosis, life expectancies, and estimated years of life lost based on sex, tumor type, and location.

Chen et al. found a decrease in incidence and lifetime risk of gastric cancer in Taiwan during the past decade. They propose that this could be because of the policy support for Helicobacter pylori eradication by the National Health Insurance in Taiwan, although further studies are needed to prove this.

Gastric cancer was more prevalent among men. The age-specific incidence rates of men are similar to those of women before 50 years of age, but men are about 1.5–2 times more likely to develop such cancer after they are 50 years old. Among the 12,403 women and 23,173 men diagnosed with gastric cancer, nearly 88.6% of patients had adenocarcinoma.

Patients with adenocarcinoma of the gastric cardia had shorter life expectancies and greater estimated years of life lost than those with non-cardia tumors. Women with gastric adenocarcinoma were diagnosed at a younger age and had longer life expectancies, but more estimated years of life lost than men with such tumors.

Importantly, Chen et al. found that when gastric adenocarcinoma was diagnosed at an early stage and cured, women had an estimated 2.62 years of life saved/case and men had 1.97 years of life saved/case.

The early detection rate for gastric cancer is higher than 40% in Japan, whereas it is just around 20% in Western countries. Chen et al. propose that increasing screening and early detection could prevent thousands of life-year loss—especially among women.

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How Does a High-Fat Diet Contribute to Pancreatic Cancer?

A high-fat diet can promote development of pancreatic cancer, but cyclooxygenase-2 (COX2) inhibitors prevent this process, according to a mouse study published in the December issue of Gastroenterology. The mechanisms appear to involve activation of oncogenic KRAS and upregulation of COX2, which promotes pancreatic inflammation.

Increased body mass index and excessive body weight are risk factors for pancreatic ductal adenocarcinoma (PDAC). This is a concern, because the number of obese individuals in the United States has doubled to 59 million during the past 2 decades. Increasing consumption of high-fat diets is contributing to the prevalence of obesity. Bincy Philip et al. investigated whether high-fat diets promote development of PDAC in mice.

Mice that express oncogenic Kras (which contains the activating G12D mutation) specifically in acinar cells (LSL-Kras/Ela-CreERT mice) develop pancreatic intraepithelial neoplasia (PanINs)—the initial step in the progression of PDAC, although at a very low rate.

The G12D mutation in KRAS is believed to be involved in the earliest stages of transformation of normal pancreatic acinar cells into PanINs. However, the mutation has been detected in a large number of healthy people who never develop PDAC, so other factors must be involved. Oncogenic KRAS is believed to require further stimuli to transform cells.

Philip et al. investigated whether the high-fat diet might be the stimulus that oncogenic KRAS needs to promote development of pancreatic cancer.

They placed LSL-Kras/Ela-CreERT mice on either control diets, in which 10% of energy comes from fat, or high-fat diets, in which 60% of energy is derived from fat.

The mice fed control diets formed a few spontaneous PanIN at young ages. However, mice fed the high-fat diets developed pancreatic inflammation and fibrosis and higher numbers of PanINs and PDACs than the mice fed control diets. The high-fat diet mice all died by 250 days, while about 70% of the mice on control diets were still alive. Pancreata of mice that did not express oncogenic Kras but were also fed high-fat diets had minor pathologic alterations.

Pancreatic tissues from LSL-Kras/Ela-CreERT mice on high-fat diets had increased levels of Kras activity than mice on control diets. The authors state that these findings support the concept that oncogenic Kras is largely inactive until stimulated, and that a high-fat diet provides the signal for full Kras activation.

High activity of Kras can promote inflammation via several mechanisms, including COX2. Philip et al. tested whether COX2 was required for high-fat diet-induced changes in pancreatic tissues.

They found that mice that expressed oncogenic Kras but had targeted deletion of COX2 in pancreatic acinar cells had no evidence of increased numbers of PanIN lesions, inflammation, or fibrosis after 30 days on high-fat diets.

Mice expressing oncogenic Kras were then placed on high-fat diets but also given celecoxib, a selective COX2 inhibitor, for 38 days. These mice had significantly lower levels of inflammation, fibrosis, and PanIN development than mice on high-fat diets given saline instead of celecoxib.

The authors conclude that a high-fat diet provides the stimulus needed to fully activate oncogenic Kras, which along with COX2 expression contributes to PDAC initiation (see below figure).

Activation of oncogenic Kras leads to activation of COX2, phosphorylation of ERK, infiltration of macrophages (blue) into the stroma, and activation of quiescent stellate cells (pink), which produce α-SMA and collagen I to promote fibrosis. COX2 is part a feedback loop that maintains Kras activity and promotes further inflammation.

Activation of oncogenic Kras leads to activation of COX2, phosphorylation of ERK, infiltration of macrophages (blue) into the stroma, and activation of quiescent stellate cells (pink), which produce α-SMA and collagen I to promote fibrosis. COX2 is part a feedback loop that maintains Kras activity and promotes further inflammation.

Philip et al. propose that COX2 is part a feedback loop that maintains Kras activity and promotes further fibrosis and inflammation in the pancreas. The findings provide insight into the connection between obesity and PDAC development, as well as interesting strategies to prevent PDAC.

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Putting a Cap on Acid Reflux

Researchers show that a polysaccharide ‘raft’ can float on top of acid in the stomach to block its backflow into the esophagus. It reduces reflux symptoms in patients with gastroesophageal reflux disease (GERD), according to a clinical trial the December issue of Clinical Gastroenterology and Hepatology.

GERD is a common condition in which backflow of stomach contents into the esophagus causes pain and often esophageal damage. Most of these acid reflux episodes occur after meals. This is because an unbuffered pool of acid floats on top of ingested food (the gastric acid pocket).

Wout O. Rohof et al. investigated whether alginates might be used to block this back-splashing of acid into the esophagus. Alginates are natural polysaccharide polymers that, on contact with gastric acid, quickly precipitate into a low-density viscous gel of near-neutral pH. The change in pH causes sodium bicarbonate in the formulation to release carbon dioxide, which floats to the top of the gastric contents like a raft—close to the esophagogastric junction, where the acid pocket forms (see video).

A video showing that the alginate-antacid remains on top of the acid pocket in the proximal stomach during the entire study period.

A video showing that the alginate-antacid remains on top of the acid pocket in the proximal stomach during the entire study period.

Rohof et al. studied the effects of a commercial alginate-antacid formulation (Gaviscon Double Action Liquid) in 16 patients with symptomatic GERD. They found that the alginate significantly reduced the number of acid reflux episodes (3.5 events), compared to patients given only an antacid after a meal (Antagel, 15 events). Furthermore, the time to acid reflux was significantly increased in patients receiving alginate-antacid (63 minutes) vs. those receiving antacid (14 minutes).

In imaging studies, the authors saw that the acid pocket was located below the diaphragm in 71% of patients given alginate-antacid vs. 21% of those given antacid. There was an inverse correlation between a sub-diaphragm position of the acid pocket and acid reflux.

Rohof et al. conclude that in patients with reflux after meals, the acid pocket can be directly targeted with a non-systemic, alginate-antacid formulation.

In an editorial that accompanies the article, Daniel Sifrim and Roberto Penagini write that the treatment is best-suited for patients with mild GERD symptoms, or possibly GERD symptoms during pregnancy. However, more data is needed to recommend alginates for patients with persistent symptoms in spite of PPI therapy.

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Are Mitochondria Involved in Ulcerative Colitis?

Changes in mitochondrial DNA that increase levels of ATP in the intestinal mucosa protect mice from colitis, according to the November issue of Gastroenterology. Strategies to increase mitochondrial ATP production by intestinal epithelial cells might therefore be developed to treat patients with ulcerative colitis (UC).

Characteristics of UC include reduced levels of ATP and disrupted energy homeostasis in the intestinal mucosa. Intestinal tissues from patients have also been shown to have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex—mitochondrial dysfunction could contribute to the pathogenesis of this inflammatory disorder.

Mitochondria

Mitochondria

Florian Bär et al. performed studies in conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex during development of colitis.

They found that mice with increased activity of the mucosal OXPHOS complex and levels of ATP (BL6.NOD and BL6.NZB mice) developed less-severe colitis after administration of dextran sodium sulfate (DSS) or trinitrobenzene sulfonate (TNBS) than mice with lower mucosal levels of ATP (BL6.BL6 and BL6.AKR mice). The BL6.NOD and BL6.NZB mice have a polymorphism in the mt-tRNAArg gene that affects tRNA function and mitochondrial biogenesis, leading to increased levels of ATP in mucosal cells.

BL6.NOD and BL6.NZB mice were almost completely protected from DSS-induced colitis, with significantly reduced disease activity index scores and smaller reductions in colon length, compared with BL6.BL6 and BL6.AKR mice. BL6.NOD and BL6.NZB mice also developed significantly less TNBS-induced colitis, with reduced weight loss and only mild endoscopic and histopathologic inflammatory changes, compared with BL6.BL6 and BL6.AKR mice.

How could the mitochondria protect against colitis? Bär et al. propose that the mitochondria mediate the epithelial regenerative potential, involving a careful balance between cell proliferation and apoptosis. These mechanisms depend on cellular energy supply and could therefore be affected by variants in mitochondrial DNA.

The authors observed significantly increased proliferation of enterocytes from BL6.NOD and BL6.NZB mice, compared with the other strains. Colon tissues from these mice also had increased activity of the transcription factor NFkB, which regulates mucosal homeostasis and epithelial cell proliferation.

In an editorial that accompanies the article, David L. Boone and Mike A. Teitell explain that mitochondria are platforms for innate immune signaling, including responses to viruses and for activation of the NLRP3 inflammasome. Further exploration of the link between variants in mtDNA, NFkB activity, intestinal cell proliferation, and protection from colitis could increase our understanding the pathogenesis of IBD.

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What is the Best Treatment for Esophageal Adenocarcioma?

Patients with esophageal adenocarcinoma (EAC) who receive endoscopic therapy survive as long as patients treated by surgery, according to the November issue of Clinical Gastroenterology and Hepatology.

Esophageal cancer is a highly fatal malignancy—approximate 19% of patients survive 5 years. The incidence of EAC, the most common form of esophageal cancer in Western countries, increased by 1.5% between 1998 and 2008. Surveillance has also increased, via endoscopy of patients with Barrett’s esophagus, leading to detection of more patients with early-stage adenocarcinoma.

These patients were formerly treated only by esophagectomy, associated with up to 10% mortality and 56% morbidity. However, endoscopic therapies such as endoscopic resection and ablation have become an effective treatment option for early-stage EAC, although it is not clear if they have been widely adopted by physicians. Furthermore, it is not clear if endoscopy and surgery are equally effective in treating patients with high-grade dysplasia and intramucosal carcinoma. Population-based data on long-term outcomes of patients with EAC after endoscopic therapy and surgery are limited.

Saowanee Ngamruengphong et al. analyzed the Surveillance Epidemiology and End Results database to determine esophageal cancer-specific survival and overall survival of patients with early EAC treated by endoscopic therapy or surgery.

They found that survival after 5 years was higher in the surgery group than in the endoscopic therapy group (70% vs 58%, respectively). However, after adjusting for patient and tumor factors, patients treated by endoscopy had similar overall survival times and esophageal cancer-specific survival times (see below figure).

Kaplan–Meier survival estimates for patients undergoing endoscopic therapy vs surgery . (A) Overall survival  and (B) esophageal cancer–specific survival.

Kaplan–Meier survival estimates for patients undergoing endoscopic therapy vs surgery . (A) Overall survival and (B) esophageal cancer–specific survival.

The authors state that patients treated by endoscopy were likely to have been at high risk for surgery, which might account for lower unadjusted overall survival in the endocscopy group.

Ngamruengphong et al. also found that the use of endoscopic therapy increased progressively from 3% in 1998 to 29% in 2009. Factors associated with use of endoscopic therapy included age older than 65 years, diagnosis in 2006−2009 vs 1998−2001, and the absence of submucosal invasion. Endoscopic and surgical therapies produced similar survival times among patients with tumor stages Tis/T1a and T1b, after adjusted multivariable regression analyses.

Endoscopic therapy was incorporated into 2012 National Comprehensive Cancer Network guidelines for esophageal cancer. For a medically fit patient with localized disease, endoscopy is the primary treatment for Tis cancer and the preferred option for T1a cancer. Esophagectomy is the primary treatment for a T1b tumor. However, endoscopic therapy is recommended for medically unfit patients with Tis and superficial T1a or T1b cancer.

The increased use of endoscopic therapy, compared with surgery, during the last decade indicates its increasing acceptance and availability; endoscopic therapy has spread from select academic centers to the general community. Ngamruengphong et al. expect its use to continue to expand.

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