Avlin B. Imaeda

Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome

Hepatocyte death results in a sterile inflammatory response that amplifies the initial insult and increases overall tissue injury. One important example of this type of injury is acetaminophen-induced liver injury, in which the initial toxic injury is followed by innate immune activation. Using mice deficient in Tlr9 and the inflammasome components Nalp3 (NACHT, LRR, and pyrin domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1, we have identified a nonredundant role for Tlr9 and the Nalp3 inflammasome in acetaminophen-induced liver injury. We have shown that acetaminophen treatment results in hepatocyte death and that free DNA released from apoptotic hepatocytes activates Tlr9. This triggers a signaling cascade that increases transcription of the genes encoding pro-IL-1beta and pro-IL-18 in sinusoidal endothelial cells. By activating caspase-1, the enzyme responsible for generating mature IL-1beta and IL-18 from pro-IL-1beta and pro-IL-18, respectively, the Nalp3 inflammasome plays a crucial role in the second step of proinflammatory cytokine activation following acetaminophen-induced liver injury. Tlr9 antagonists and aspirin reduced mortality from acetaminophen hepatotoxicity. The protective effect of aspirin on acetaminophen-induced liver injury was due to downregulation of proinflammatory cytokines, rather than inhibition of platelet degranulation or COX-1 inhibition. In summary, we have identified a 2-signal requirement (Tlr9 and the Nalp3 inflammasome) for acetaminophen-induced hepatotoxicity and some potential therapeutic approaches.

Cell Death and Fibrogenesis

Fibrosis is a common feature of chronic liver injury and is initiated by cell death inside the liver. Hepatocyte death results in apoptotic bodies and other cellular debris, which are phagocytosed by hepatic stellate cells (HSCs), resulting in their activation, proliferation, differentiation, and matrix deposition. This profibrotic effect of cellular death is balanced by an antifibrotic effect of HSC death. Many HSC survival signals are obtained from the extracellular matrix, and active proapoptotic signals are provided by immune cells, particularly natural killer (NK) cells. Quiescent HSCs are relatively resistant to apoptotic signals but become sensitive after activation. The important role of NK cells in inducing HSC apoptosis may explain the increased fibrosis associated with immune suppression (e.g., in the transplant recipient) and HIV infection. HSCs also undergo senescence, which limits their function and sensitizes them to apoptosis.

What Is Most Important to Patients when Deciding about Colorectal Screening

BACKGROUNDColorectal cancer (CRC) screening can be administered through tests with varied characteristics and is a preference-sensitive decision.OBJECTIVETo assess patient experiences with a Maximum Differences Scaling (MDS) tool for eliciting values about CRC screening test characteristics and determine whether patients vary in how they prioritize test characteristics and whether this variation relates to test preferences.DESIGNMDS survey to elicit patients’ values for characteristics related to fecal occult blood testing, sigmoidoscopy, colonoscopy, CT colonography and colon capsule endoscopy.PARTICIPANTS92 patients enrolled in primary care clinics at a VA hospital and associated university.RESULTSPatients reported that the tool was easy to use (95%). On completion 62% would choose colonoscopy, 23% colon capsule endoscopy and 10% CT colonography. Of the attributes evaluated, patients valued sensitivity, risk of tear and need for a second test most. Sensitivity was more important to those choosing colonoscopy than those choosing other tests (median importance = 21.5 versus 19.6, p < 0.01). Concern with complications and sedation was positively associated with age (p < 0.001 and p < 0.001), whereas concern with colon preparation and missing work was negatively associated with age (p < 0.009 and p < 0.03). Patients with fair or poor health status were less concerned with sensitivity than patients in good to excellent health (median importance = 19.3 versus 21.4, p < 0.008).CONCLUSIONSThis pilot study suggests that patients vary in how they prioritize colorectal cancer screening test attributes; this variation is associated with test preferences, and this MDS tool is feasible to use and may help patients construct their preferences.

Small Intestinal Angioectasias Are Not Randomly Distributed in the Small Bowel and Most May Be Reached by Push Enteroscopy.

Goals: The goal was to describe the location of angioectasias within small bowel on capsule endoscopy and the utility of push enteroscopy versus deep enteroscopy in treatment of overt bleeding from these. Background: Overt bleeding from small bowel angioectasias is a clinical challenge. Thalidomide and octreotide can be difficult to prescribe and may not be effective. Endoscopy remains a mainstay of treatment for overt bleeding from angioectasias but data regarding the long-term efficacy of endoscopic therapy are limited. We sought to define the location of small bowel angioectasias using capsule endoscopy and review our outcomes for push and double-balloon enteroscopy. Methods: We retrospectively reviewed all 428 capsule endoscopy studies from the Veterans Administration Hospital in West Haven, CT from 2005 to 2012. Location of angioectasias was evaluated using lead mapping and small bowel transit time. Results: We identified 69 patients with small bowel angioectasia. At least 66.8% of lesions were in duodenum or ligament of Treitz, with 78.3% within the first 25% of small bowel transit. Twenty-four patients underwent endoscopic treatment of overt bleeding from small bowel angioectasias. Thirty-three percent rebled requiring multiple procedures. Eight patients had 10 anterograde double-balloon examinations. Only 2 patients had both cessation of bleeding with double balloon and lesions outside the reach of push enteroscopy. Conclusions: Push enteroscopy for bleeding angioectasias is effective in many patients. Deep enteroscopy may not benefit most patients as most angioectasias are proximal. Patients with deeper lesions tended to have multiple lesions and rebleeding despite deep enteroscopy.

Preclinical endoscopic training using a part-task simulator: learning curve assessment and determination of threshold score for advancement to clinical endoscopy

BackgroundPreclinical simulator training has the potential to decrease endoscopic procedure time and patient discomfort. This study aims to characterize the learning curve of endoscopic novices in a part-task simulator and propose a threshold score for advancement to initial clinical cases.MethodsTwenty novices with no prior endoscopic experience underwent repeated endoscopic simulator sessions using the part-task simulator. Simulator scores were collected; their inverse was averaged and fit to an exponential curve. The incremental improvement after each session was calculated. Plateau was defined as the session after which incremental improvement in simulator score model was less than 5%. Additionally, all participants filled out questionnaires regarding simulator experience after sessions 1, 5, 10, 15, and 20. A visual analog scale and NASA task load index were used to assess levels of comfort and demand.ResultsTwenty novices underwent 400 simulator sessions. Mean simulator scores at sessions 1, 5, 10, 15, and 20 were 78.5 ± 5.95, 176.5 ± 17.7, 275.55 ± 23.56, 347 ± 26.49, and 441.11 ± 38.14. The best fit exponential model was [time/score] = 26.1 × [session #]−0.615; r2 = 0.99. This corresponded to an incremental improvement in score of 35% after the first session, 22% after the second, 16% after the third and so on. Incremental improvement dropped below 5% after the 12th session corresponding to the predicted score of 265. Simulator training was related to higher comfort maneuvering an endoscope and increased readiness for supervised clinical endoscopy, both plateauing between sessions 10 and 15. Mental demand, physical demand, and frustration levels decreased with increased simulator training.ConclusionPreclinical training using an endoscopic part-task simulator appears to increase comfort level and decrease mental and physical demand associated with endoscopy. Based on a rigorous model, we recommend that novices complete a minimum of 12 training sessions and obtain a simulator score of at least 265 to be best prepared for clinical endoscopy.

467 Aspirin Blocks Acetaminophen Induced Hepatotoxicity and Mortality in Mice - Dependent On the ASC/Caspase-1 Inflammasome

Acetaminophen (APAP) hepatotoxicity is dependent on the immune system, but the responsible molecular pathways have not been identified. Recently it was reported that IL-1 receptor signaling is required for APAP induced hepatotoxicity (Nature Med. July 2007, vol. 13,7. p851). The protein complex of pattern recognition molecules, the adaptor protein ASC and caspase-1 constitutes the inflammasome. Activation of the inflammasome results in production of IL-1 beta, resulting in IL-1 receptor signaling. Aims: i) Determine the role of the inflammasome in APAP hepatotoxicity. ii) Test if aspirin down-modulates the inflammasome pathway. iii) Test if aspirin blocks APAP hepatotoxicity. Methods: APAP hepatotoxicity (500 mg/kg ip single injection) was induced in wild-type C57BL/6 mice, and mice deficient in inflammasome components ASC, or caspase-1. An In-Vivo assay of inflammation dependent on the ASC/Caspase-1 pathway was used to test if aspirin can reduce inflammation mediated by this pathway. This consisted of intraperitoneal (ip) injection of monosodium urate (MSU) (3mg/mouse), with quantification of peritoneal neutrophils at 3 hrs. The ability of aspirin (approx 6mg/kg in drinking water starting 3 days prior to APAP), the platelet inhibitor clopidogrel (30 mg/kg by gavage every 24 hrs starting 2 days prior to APAP) and a cox-1 inhibitor (SC-560, 5mg/kg by gavage every 12 hrs starting 60 hrs before APAP) to protect from APAP induced injury was tested. Results: Mice deficient in the adaptor protein ASC or caspase-1 had reduced mortality from hepatotoxic doses of APAP (P<0.027 and P<0.013 respectively). In wild-type mice aspirin reduced inflammation mediated by the ASC/Caspase1 inflammasome as demonstrated by reduced ip neutrophil accumulation (P<0.015). In wild-type mice at 12 hrs after APAP injection aspirin improved histology and transaminitis (control group mean ALT 2365 iu/ml SD 1801 vs aspirin group mean ALT 944 iu/ml SD 373 p<0.04). In wild-type mice aspirin also reduced mortality from APAP hepatotoxicity (at 72 hrs after APAP mortality was 10/13 in control group and 3/17 in group receiving aspirin P<0.015). Clopidogrel and the cox-1 inhibitor SC-560 did not reduce mortality from APAP hepatotoxicity, suggesting that the mechanism of action of aspirin was not via inhibition of platelet degranulation or cox-1. Conclusions: The ASC/Caspase-1 inflammasome is required for APAP induced hepatotoxicity, and can be suppressed by aspirin. Aspirin reduces APAP induced mortality. Co-formulation of aspirin with APAP may reduce APAP induced liver failure.

Confocal laser endomicroscopy for the detection of atrophic gastritis: a new application for confocal endomicroscopy?

New technologies in the world of endoscopy abound, but success and widespread use is not always assured. Technologies that fill an unmet need or improve upon current technology in widespread use are more likely to be successful. Confocal laser endomicroscopy (CLE) is a technology that allows the user to get microscopic views of the mucosa in real time during endoscopy. The technology can be used through a single endoscope-based system (CLE) (OptiScan, Notting Hill, Australia) or through a probe-based system known as probe-based confocal laser endomicroscopy (PCLE) (Cellvizio; Mauna Kea Technologies, Paris, France). These technologies differ somewhat in the depth of penetration, variable with the endoscopebased system, and fixed with the probe-based system, and the size of the field of view. Both rely on fluorescence. In tissues without autofluorescence, as is the case for most mucosal tissues, a dye must be administered either topically or intravenously (fluorescein). Confocal technology has been applied for several uses throughout the GI tract, typically to distinguish neoplastic from non-neoplastic tissue, but also to diagnose and stage inflammatory conditions. Some applications are relatively well established in terms of evidence and clinical use; some applications have supportive data but are rarely used clinically, and new applications that may fit unmet clinical needs are being studied. Perhaps the most promoted use of endoscopic confocal technology has been in Barrett’s esophagus surveillance. A randomized controlled trial showed a higher diagnostic yield for patients randomized to highdefinition white light endoscopy and CLE with targeted biopsies than for standard highdefinition white light endoscopy and random biopsies.1 Narrow band imaging (NBI) was not compared in this study. The detection of neoplasia was comparable; the CLE group simply required fewer biopsies for diagnosis. This study, as in many studies using this advanced technology, was performed by expert endoscopists limiting the generalizability of the results. In addition, surveillance of large areas of involved mucosa is limited by the ability to visualize only tiny areas at a time, such that screening a long segment would be unwieldy and still require operator selection of areas to evaluate. This is likely more time consuming than targeted biopsy using high-definition white light and NBI. PCLE has been used to evaluate biliary strictures and suspected cholangiocarcinomas. Because cholangiocarcinoma can be otherwise difficult to diagnose, this is an area ripe for effective technology.2 CLE and PCLE have been used to distinguish neoplastic from non-neoplastic polyps in the colon. Some studies show sensitivity and specificity >90%, suggesting that a strategy to diagnose and discard could be reasonable.3,4 However, 1 study showed only moderate interobserver agreement for distinguishing neoplastic from non-neoplastic lesions.5 The technology has been used to detect flat dysplasia and disease activity in patients with inflammatory bowel disease and to diagnose microscopic colitis and celiac disease.6–8 These uses of confocal technology are less commonly applied in clinical use. CLE has been reported to have some amount of success in diagnosing gastric neoplasia and early gastric cancer, but lesions along the incisura may be difficult to examine because of limited flexibility of the endoscope.9–11 More recently it was used to detect mucosal responses to food antigens in patients with irritable bowel syndrome and food intolerances.12,13 PCLE is being used to evaluate pancreatic cysts, and a probe through which a biopsy needle may be passed is being evaluated. These new uses of the technology may gain broader acceptance as they fill unmet clinical needs. In any case, endoscopic confocal technology has not yet gained widespread use. Cost, reimbursement, and required additional time are limiting. As noted above, PCLE and the endoscopic confocal have limited fields of

Unsuspected Colonic Hemorrhage Found on Screening Colonoscopy

Section of Digestive Diseases, Section of General Internal Medicine, Yale University School of Medicine, New Haven, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 Question: A 68-year-old man with hypertension, multiple sclerosis, and neurogenic bladder underwent an inpatient bowel cleansing owing to immobility for a routine screening colonoscopy. The patient was asymptomatic, had no complaints, and no other activemedical issues at the time of preparation and procedure. He denied any nausea, vomiting, abdominal pain, changes in bowelmovements, bright red blood per rectum, andmelena. The patient denied recent changes in his weight. He denied any family history of inflammatory bowel disease, colon cancer, and other gastrointestinal malignancies. His medications included interferon, primidone, furosemide, ferrous sulfate, Senna, docusate, and subcutaneous heparin, which was started during the admission. On physical examination, hewas a thinman, afebrile with a temperature of 36.8 C, blood pressurewas 150/90mm Hg, and pulse rate was 75 beats per minute. His abdomen was flat, soft, nondistended, and nontender with no hepatosplenomegaly appreciated. Admission bloodwork the day before colonoscopy revealed a normalwhite blood cell count, hemoglobin 13.4 g/dL (normal, 12.8-17), mean corpuscular volume 94.6 mm (normal, 82-99), and platelet count of 146 10/mL (normal, 140-360 10/mL). His chemistries were within normal limits. International Normalized Ratio was 1 (normal, 0.9-1.1). A split dose bowel cleansing regimen was used and the patient completed his bowel cleansing solution (consisting of 2 L polyethylene glycol-3350 solution in conjunction with 300 mL of magnesium citrate) in the morning of his scheduled colonoscopy and had nonbloody bowel movements throughout. He and his nurse both reported that he had a clear liquid bowel movement at 6 AM on the morning of the procedure and no subsequent bowel movements. On the day of the examination, the patient was asymptomatic and appeared well with normal vital signs. On entry into the rectum, there was a large amount of dark red blood (Figure A). The lesion shown was identified in the cecum (Figure B). No blood was seen in the ileum. Figure C shows the external abdomen. What is the diagnosis? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. 111 112 113 114 115 116 Conflicts of interest The authors disclose no conflicts.