- Email: [email protected]
Mitigation of Pruritus during Obeticholic Acid Treatment in Patients with Primary Biliary Cirrhosis: Strategies and Successes
POSTER PRESENTATIONS mice. BDL mice also demonstrated increased hippocampal CA3 regional area (ratio of CA3 region to whole brain; BDR: 0.013 ± 0.005 vs sham: 0.010 ± 0.004; n = 9 and 8 mice/grp; p < 0.04; see Figure), without a significant increase in Mn2+ – associated SI, suggesting increased CA3 neural connectivity/neuronal branching to areas close to CA3 in BDL mice. The hippocampal CA3 region is important in regulating cognition and emotional memory. Interestingly, activation of neuronal projections from the amygdala to the CA3 region have previously been associated with social withdrawal and reduced mobility in animal models; behavioral changes previously documented in BDL mice and reported by cholestatic patients.
Conclusions: MEMRI detects regionally specific brain enhancement changes in BDL vs sham mice. These findings indicate that behavioral changes documented previously in cholestatic mice are associated with brain region-specific changes in neural activity and may help explain changes in brain function (e.g. emotional regulation, cognition and reduced social interactions) that commonly occur in the setting of cholestatic liver disease. SAT-356 NEUROIMAGING EVIDENCE OF HIPPOCAMPAL CHANGES IN PRIMARY BILIARY CIRRHOSIS CONSISTENT WITH TISSUE INJURY OR STRESS V. Mosher1, M.G. Swain2, Glenda MacQueen3, Bradley G. Goodyear4. 1 Seaman Family MR Research Centre; 2Liver Unit - Calgary Division of Gastroenterology and Hepatology; 3Psychiatry; 4Seaman Family MR Centre, University of Calgary, Calgary, Canada E-mail: [email protected] Background and Aims: Patients with Primary Biliary Cirrhosis (PBC) commonly experience symptoms which adversely affect their quality of life and include altered mood and decreased cognition. The hippocampus is strongly associated with memory and mood control. Quantitative susceptibility mapping (QSM) is a novel magnetic resonance imaging (MRI) technique that uses localized magnetic field inhomogeneities within tissue during MRI (i.e. increased susceptibility due to iron or deoxyheme deposition, or demyelination) and can be used to infer tissue injury or oxidative stress. Therefore, we hypothesized that PBC patients would exhibit increased susceptibility within the hippocampus. Methods: Seven female PBC patients and seven female healthy controls underwent MRI using a 3 Tesla General Electric MR scanner. All PBC patients were non-cirrhotic. MR images were collected with pixel intensity sensitive to susceptibility. QSM images were created using an in-house custom program (Cerebra-QSM; Calgary Image Processing and Analysis Centre) and registered to a standard brain template to permit the comparison between patients and controls on a pixel-by-pixel basis using a General Linear Model. Results: As shown in the Figure, increased susceptibility was observed bilaterally within the hippocampus as well as within the
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left thalamus of the group of PBC patients as compared to the group of healthy controls (red colour). Decreased susceptibility (blue colour) was observed bilaterally within the globus pallidus of the basal ganglia in the group of PBC patients (a brain area important for motivational behavior and fatigue).
Conclusions: Increased susceptibility within the hippocampus and thalamus of PBC patients suggest tissue injury or oxidative stress, and may be linked to disturbances in cognition and mood seen in PBC patients. The thalamus is critical for relaying sensory information from the body to the brain, and increased susceptibility therefore may underlie altered processing of peripheral signals to the brain in PBC, which in turn may be linked to altered brain function. The reason for decreased susceptibility within the globus pallidus is unknown, but could possibly be linked to the development of fatigue in PBC patients and warrants further investigation. Full neuropsychological assessment of this cohort were performed and are being analyzed to fully establish the utility of QSM for elucidating the causes of altered brain function (i.e. cognition, mood impairments, and fatigue) observed in PBC. SAT-357 MITIGATION OF PRURITUS DURING OBETICHOLIC ACID TREATMENT IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS: STRATEGIES AND SUCCESSES M.J. Mayo1, A. Kremer2, U. Beuers3, T. Marmon4, R. Hooshmand-Rad4, R. Pencek4, D. Shapiro4. 1UT Southwestern Medical Center, Dallas, United States; 2Department of Medicine, Friedrich-Alexander-University, Erlangen, Germany; 3Department of Gastroenterology and Hepatology, AMC, Amsterdam, Netherlands; 4Intercept Pharmaceuticals, Inc., San Diego, United States E-mail: [email protected] Background and Aims: Obeticholic acid (OCA), a potent, selective FXR agonist significantly improved liver biochemistry in 3 randomized, placebo (PBO)-controlled trials. Pruritus, a common symptom of primary biliary cirrhosis (PBC), was the most common adverse event (AE) across the double-blind trials. Based on Phase 2 observations of dose related increases in pruritus, and comparable efficacy with OCA doses ≥10 mg, a Phase 3 trial (POISE) assessed a 10 mg dose and a 5 mg dose with the option to titrate to 10 mg after 6 mo based on response and tolerability (TITR arm). Other strategies to mitigate pruritus included dose interruption, decreased dosing frequency and concomitant medications. Methods: 216 PBC patients (alkaline phosphatase [ALP] ≥1.67× ULN or total bilirubin >ULN but <2× ULN) were randomized 1:1:1 to PBO, TITR, or OCA 10 mg and dosed. Pretrial ursodeoxycholic acid was to be continued. Strategies to mitigate pruritus included: dose frequency
Journal of Hepatology 2016 vol. 64 | S631–S832
POSTER PRESENTATIONS changes or drug holidays (<28 days), and use of medications like bile acid sequestrants (BAS). Physicians recorded AEs of pruritus including severity and medication use at each clinic visit. Results: Mild to moderate pruritus was the most common AE in POISE (38%, 56%, and 70% in the PBO, TITR, and OCA 10 mg groups); this led to discontinuation of drug in 0%, 1% and 10% in the PBO, TITR, and OCA 10 mg groups, respectively). Titration improved tolerability of pruritus without compromising efficacy. After 12 mo treatment, ALP reduction was similar between TITR and OCA 10 mg. In the subset of patients who experienced pruritus, titration delayed both time to onset of pruritus (median days: PBO, 51; TITR, 24; OCA 10 mg, 9) and reduced severity of pruritus compared to those in the 10 mg group (% patients with severe pruritus: PBO, 7%; TITR, 15% of patients who titrated to 10 mg [n = 33]; OCA 10 mg, 23%). Many patients who experienced pruritus did not require an intervention: 50%, 38%, and 41% of the PBO, TITR, and OCA 10 mg groups. While clinically meaningful improvements in ALP were still observed with concomitant BAS use, efficacy was modestly attenuated at OCA 5 mg but not impacted at 10 mg. Conclusions: In PBC patients administered OCA, pruritus can be managed successfully. Initiation of OCA at 5 mg followed by an increase to 10 mg resulted in a lower incidence of pruritus over 12 mo, reduced severity of pruritus, and reduced OCA discontinuations due to pruritus without compromising efficacy. Guideline-approved standard of care for cholestatic pruritus can also be applied. SAT-358 MODULATION OF THE TRANSCRIPTION FACTORS SNAIL, SLUG AND TWIST IN RESPONSE TO BILIARY INJURY IN PRIMARY BILIARY CIRRHOSIS M. Jan1, J. Kirby1, J. Brain1, Applied Immunobiology and Transplantation Research Group. 1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Primary biliary cirrhosis (PBC) is an autoimmune liver disease leading to ductopenia and fibrosis. Loss of epithelial cell function is key to this process. Oxidative stress, the result of a pathogenic inflammatory process, may induce epithelial to mesenchymal transition (EMT). The transcription factors snail, slug and twist have been implicated during EMT in other tissues. It is not known whether these transcription factors play a role in PBC. Aim: To define the molecular mechanism by which oxidative stress induces EMT in bile ducts. Methods: The immortalised biliary epithelial cell (BEC) line H69 was treated with hydrogen peroxide (200 μM for 2 hours) to model oxidative stress. Enzyme-linked immunosorbent assay (ELISA) was used to detect TGFβ expression. Real-time PCR was used to determine mRNA expression of snail, slug and twist; proteomic features of EMT were confirmed by immunohistochemistry. The TGFβ receptor antagonist SB-505124 was used to study the effects of inhibition of TGFβ on the process of oxidative stress-induced EMT, and the induction of snail, slug and twist. Results: Oxidative stress induced the expression of TGFβ2, and initiated EMT in BEC; with con-concomitant upregulation of snail, slug and twist. SB-505124 pre-treatment inhibited EMT, prevented upregulation of snail, slug and twist, and conserved the phenotype of functional BEC. Conclusions: Oxidative stress upregulates snail, slug and twist expression in BEC by a mechanism involving autocrine TGFβ2. Blockade of the TGFβ receptor inhibits EMT in this model of PBC, suggesting potentiality as biomarkers of disease progression and a possible therapeutic modality.
SAT-359 FEMALE PREDOMINANCE, LOW RATE OF PRIMARY SCLEROSING CHOLANGITIS-IBD PHENOTYPE, LOW RATE OF DOMINANT STRICTURES, CHOLANGIOCARCINOMA AND NEED FOR TRANSPLANTATION M. Lita1, S. Iacob1, R. Iacob1, C. Ester1, R. Cerban1, L. Gheorghe1. 1 Gastroenterology and Hepatology, Fundeni Clinical Institute, Bucharest, Romania E-mail: [email protected] Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease involving intra- and extrahepatic biliary ducts, characterized by progressive sclerosis and destruction of the biliary system. The course of the disease is often complicated by biliary strictures and cholangiocarcinoma. However, it is unknown if phenotypic differences exist among patients diagnosed with PSC in different populations. To this aim, we compared the clinical characteristics of a PSC cohort in a large-volume Romanian center for liver disease and liver transplantation with literature data. Methods: The study was a single – center prospective cohort study that included all consecutive patients diagnosed with primary sclerosing cholangitis admitted in our center between January 1, 2011 and November 1, 2015. Clinical, biochemical, histological and imaging data were analyzed. Results: From a total of 61 patients diagnosed with primary sclerosing cholangitis, 62.29% were female and 37.71% males, with an average age at diagnosis of 43.59 ± 16.34 years. In 8.19% of cases (5 patients) was established the diagnosis of small bile ducts primary sclerosing cholangitis and 11.47% of cases (7 patients) were diagnosed with primary sclerosing cholangitis – autoimmune hepatitis overlap syndrome. Most common, the diagnosis was established using magnetic resonance techniques, in 68% of cases, and in 32% of cases liver biopsy was performed. A number of 6 patients (9.83%) were diagnosed with dominant strictures. Cholangiocarcinoma was established in 2 patients (3.27%). A number of 17 patients (27.86%) were included on the waiting list and for 7 patients (11.47%) liver transplantation was necessary. PSC recurrence after liver transplantation occurred in 2 cases (28.57%). Association with inflammatory bowel disease was observed in 21.31% of cases (13 patients), 8 of them with ulcerative colitis (61.53%). Conclusions: In a tertiary gastroenterology center, primary sclerosing cholangitis is a rare pathology, with middle age onset, predominant in female patients, with low rates of complications due to dominant biliary strictures or cholangiocarcinoma and low need for liver transplantation. Association with inflammatory bowel disease is rare, with the predominance of ulcerative colitis. SAT-360 LOSS OF CELLULAR FLICE-INHIBITORY PROTEIN PROMOTES CHOLESTASIS-INDUCED HEPATOCELLULAR INJURY AND INFLAMMATION IN BILE DUCT LIGATED MICE N. Gehrke1, Y. Alt1, M. Nagel1, M. Schuchmann2, P.R. Galle1, J.M. Schattenberg1. 1I. Department of Medicine, University Medical Center, Johannes Gutenberg-University Mainz, Mainz; 2I. Department of Medicine, Klinikum Konstanz, Konstanz, Germany E-mail: [email protected] Background and Aims: Cholestasis, which is characterized by an abnormal accumulation of toxic bile acids in the liver due to impairment of bile flow, causes hepatocellular injury and fibrosis. Toxic bile acids-induced hepatocyte apoptosis contributes to these processes. The aim of this study was to evaluate the influence of the anti-apoptotic caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) on acute cholestatic liver injury. Methods: 8–12 week old mice exhibiting a hepatocyte-specific deletion of cFLIP using albumin driven cre-expression (Alb-Cre: FLIPf/f, cFLIP−/−) and wild type littermates were examined in a bile duct ligation (BDL) model of cholestasis. Therefore a laparotomy was performed in anesthetized mice and the common bile duct was
Journal of Hepatology 2016 vol. 64 | S631–S832
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Conclusions: MEMRI detects regionally specific brain enhancement changes in BDL vs sham mice. These findings indicate that behavioral changes documented previously in cholestatic mice are associated with brain region-specific changes in neural activity and may help explain changes in brain function (e.g. emotional regulation, cognition and reduced social interactions) that commonly occur in the setting of cholestatic liver disease. SAT-356 NEUROIMAGING EVIDENCE OF HIPPOCAMPAL CHANGES IN PRIMARY BILIARY CIRRHOSIS CONSISTENT WITH TISSUE INJURY OR STRESS V. Mosher1, M.G. Swain2, Glenda MacQueen3, Bradley G. Goodyear4. 1 Seaman Family MR Research Centre; 2Liver Unit - Calgary Division of Gastroenterology and Hepatology; 3Psychiatry; 4Seaman Family MR Centre, University of Calgary, Calgary, Canada E-mail: [email protected] Background and Aims: Patients with Primary Biliary Cirrhosis (PBC) commonly experience symptoms which adversely affect their quality of life and include altered mood and decreased cognition. The hippocampus is strongly associated with memory and mood control. Quantitative susceptibility mapping (QSM) is a novel magnetic resonance imaging (MRI) technique that uses localized magnetic field inhomogeneities within tissue during MRI (i.e. increased susceptibility due to iron or deoxyheme deposition, or demyelination) and can be used to infer tissue injury or oxidative stress. Therefore, we hypothesized that PBC patients would exhibit increased susceptibility within the hippocampus. Methods: Seven female PBC patients and seven female healthy controls underwent MRI using a 3 Tesla General Electric MR scanner. All PBC patients were non-cirrhotic. MR images were collected with pixel intensity sensitive to susceptibility. QSM images were created using an in-house custom program (Cerebra-QSM; Calgary Image Processing and Analysis Centre) and registered to a standard brain template to permit the comparison between patients and controls on a pixel-by-pixel basis using a General Linear Model. Results: As shown in the Figure, increased susceptibility was observed bilaterally within the hippocampus as well as within the
S636
left thalamus of the group of PBC patients as compared to the group of healthy controls (red colour). Decreased susceptibility (blue colour) was observed bilaterally within the globus pallidus of the basal ganglia in the group of PBC patients (a brain area important for motivational behavior and fatigue).
Conclusions: Increased susceptibility within the hippocampus and thalamus of PBC patients suggest tissue injury or oxidative stress, and may be linked to disturbances in cognition and mood seen in PBC patients. The thalamus is critical for relaying sensory information from the body to the brain, and increased susceptibility therefore may underlie altered processing of peripheral signals to the brain in PBC, which in turn may be linked to altered brain function. The reason for decreased susceptibility within the globus pallidus is unknown, but could possibly be linked to the development of fatigue in PBC patients and warrants further investigation. Full neuropsychological assessment of this cohort were performed and are being analyzed to fully establish the utility of QSM for elucidating the causes of altered brain function (i.e. cognition, mood impairments, and fatigue) observed in PBC. SAT-357 MITIGATION OF PRURITUS DURING OBETICHOLIC ACID TREATMENT IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS: STRATEGIES AND SUCCESSES M.J. Mayo1, A. Kremer2, U. Beuers3, T. Marmon4, R. Hooshmand-Rad4, R. Pencek4, D. Shapiro4. 1UT Southwestern Medical Center, Dallas, United States; 2Department of Medicine, Friedrich-Alexander-University, Erlangen, Germany; 3Department of Gastroenterology and Hepatology, AMC, Amsterdam, Netherlands; 4Intercept Pharmaceuticals, Inc., San Diego, United States E-mail: [email protected] Background and Aims: Obeticholic acid (OCA), a potent, selective FXR agonist significantly improved liver biochemistry in 3 randomized, placebo (PBO)-controlled trials. Pruritus, a common symptom of primary biliary cirrhosis (PBC), was the most common adverse event (AE) across the double-blind trials. Based on Phase 2 observations of dose related increases in pruritus, and comparable efficacy with OCA doses ≥10 mg, a Phase 3 trial (POISE) assessed a 10 mg dose and a 5 mg dose with the option to titrate to 10 mg after 6 mo based on response and tolerability (TITR arm). Other strategies to mitigate pruritus included dose interruption, decreased dosing frequency and concomitant medications. Methods: 216 PBC patients (alkaline phosphatase [ALP] ≥1.67× ULN or total bilirubin >ULN but <2× ULN) were randomized 1:1:1 to PBO, TITR, or OCA 10 mg and dosed. Pretrial ursodeoxycholic acid was to be continued. Strategies to mitigate pruritus included: dose frequency
Journal of Hepatology 2016 vol. 64 | S631–S832
POSTER PRESENTATIONS changes or drug holidays (<28 days), and use of medications like bile acid sequestrants (BAS). Physicians recorded AEs of pruritus including severity and medication use at each clinic visit. Results: Mild to moderate pruritus was the most common AE in POISE (38%, 56%, and 70% in the PBO, TITR, and OCA 10 mg groups); this led to discontinuation of drug in 0%, 1% and 10% in the PBO, TITR, and OCA 10 mg groups, respectively). Titration improved tolerability of pruritus without compromising efficacy. After 12 mo treatment, ALP reduction was similar between TITR and OCA 10 mg. In the subset of patients who experienced pruritus, titration delayed both time to onset of pruritus (median days: PBO, 51; TITR, 24; OCA 10 mg, 9) and reduced severity of pruritus compared to those in the 10 mg group (% patients with severe pruritus: PBO, 7%; TITR, 15% of patients who titrated to 10 mg [n = 33]; OCA 10 mg, 23%). Many patients who experienced pruritus did not require an intervention: 50%, 38%, and 41% of the PBO, TITR, and OCA 10 mg groups. While clinically meaningful improvements in ALP were still observed with concomitant BAS use, efficacy was modestly attenuated at OCA 5 mg but not impacted at 10 mg. Conclusions: In PBC patients administered OCA, pruritus can be managed successfully. Initiation of OCA at 5 mg followed by an increase to 10 mg resulted in a lower incidence of pruritus over 12 mo, reduced severity of pruritus, and reduced OCA discontinuations due to pruritus without compromising efficacy. Guideline-approved standard of care for cholestatic pruritus can also be applied. SAT-358 MODULATION OF THE TRANSCRIPTION FACTORS SNAIL, SLUG AND TWIST IN RESPONSE TO BILIARY INJURY IN PRIMARY BILIARY CIRRHOSIS M. Jan1, J. Kirby1, J. Brain1, Applied Immunobiology and Transplantation Research Group. 1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Primary biliary cirrhosis (PBC) is an autoimmune liver disease leading to ductopenia and fibrosis. Loss of epithelial cell function is key to this process. Oxidative stress, the result of a pathogenic inflammatory process, may induce epithelial to mesenchymal transition (EMT). The transcription factors snail, slug and twist have been implicated during EMT in other tissues. It is not known whether these transcription factors play a role in PBC. Aim: To define the molecular mechanism by which oxidative stress induces EMT in bile ducts. Methods: The immortalised biliary epithelial cell (BEC) line H69 was treated with hydrogen peroxide (200 μM for 2 hours) to model oxidative stress. Enzyme-linked immunosorbent assay (ELISA) was used to detect TGFβ expression. Real-time PCR was used to determine mRNA expression of snail, slug and twist; proteomic features of EMT were confirmed by immunohistochemistry. The TGFβ receptor antagonist SB-505124 was used to study the effects of inhibition of TGFβ on the process of oxidative stress-induced EMT, and the induction of snail, slug and twist. Results: Oxidative stress induced the expression of TGFβ2, and initiated EMT in BEC; with con-concomitant upregulation of snail, slug and twist. SB-505124 pre-treatment inhibited EMT, prevented upregulation of snail, slug and twist, and conserved the phenotype of functional BEC. Conclusions: Oxidative stress upregulates snail, slug and twist expression in BEC by a mechanism involving autocrine TGFβ2. Blockade of the TGFβ receptor inhibits EMT in this model of PBC, suggesting potentiality as biomarkers of disease progression and a possible therapeutic modality.
SAT-359 FEMALE PREDOMINANCE, LOW RATE OF PRIMARY SCLEROSING CHOLANGITIS-IBD PHENOTYPE, LOW RATE OF DOMINANT STRICTURES, CHOLANGIOCARCINOMA AND NEED FOR TRANSPLANTATION M. Lita1, S. Iacob1, R. Iacob1, C. Ester1, R. Cerban1, L. Gheorghe1. 1 Gastroenterology and Hepatology, Fundeni Clinical Institute, Bucharest, Romania E-mail: [email protected] Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease involving intra- and extrahepatic biliary ducts, characterized by progressive sclerosis and destruction of the biliary system. The course of the disease is often complicated by biliary strictures and cholangiocarcinoma. However, it is unknown if phenotypic differences exist among patients diagnosed with PSC in different populations. To this aim, we compared the clinical characteristics of a PSC cohort in a large-volume Romanian center for liver disease and liver transplantation with literature data. Methods: The study was a single – center prospective cohort study that included all consecutive patients diagnosed with primary sclerosing cholangitis admitted in our center between January 1, 2011 and November 1, 2015. Clinical, biochemical, histological and imaging data were analyzed. Results: From a total of 61 patients diagnosed with primary sclerosing cholangitis, 62.29% were female and 37.71% males, with an average age at diagnosis of 43.59 ± 16.34 years. In 8.19% of cases (5 patients) was established the diagnosis of small bile ducts primary sclerosing cholangitis and 11.47% of cases (7 patients) were diagnosed with primary sclerosing cholangitis – autoimmune hepatitis overlap syndrome. Most common, the diagnosis was established using magnetic resonance techniques, in 68% of cases, and in 32% of cases liver biopsy was performed. A number of 6 patients (9.83%) were diagnosed with dominant strictures. Cholangiocarcinoma was established in 2 patients (3.27%). A number of 17 patients (27.86%) were included on the waiting list and for 7 patients (11.47%) liver transplantation was necessary. PSC recurrence after liver transplantation occurred in 2 cases (28.57%). Association with inflammatory bowel disease was observed in 21.31% of cases (13 patients), 8 of them with ulcerative colitis (61.53%). Conclusions: In a tertiary gastroenterology center, primary sclerosing cholangitis is a rare pathology, with middle age onset, predominant in female patients, with low rates of complications due to dominant biliary strictures or cholangiocarcinoma and low need for liver transplantation. Association with inflammatory bowel disease is rare, with the predominance of ulcerative colitis. SAT-360 LOSS OF CELLULAR FLICE-INHIBITORY PROTEIN PROMOTES CHOLESTASIS-INDUCED HEPATOCELLULAR INJURY AND INFLAMMATION IN BILE DUCT LIGATED MICE N. Gehrke1, Y. Alt1, M. Nagel1, M. Schuchmann2, P.R. Galle1, J.M. Schattenberg1. 1I. Department of Medicine, University Medical Center, Johannes Gutenberg-University Mainz, Mainz; 2I. Department of Medicine, Klinikum Konstanz, Konstanz, Germany E-mail: [email protected] Background and Aims: Cholestasis, which is characterized by an abnormal accumulation of toxic bile acids in the liver due to impairment of bile flow, causes hepatocellular injury and fibrosis. Toxic bile acids-induced hepatocyte apoptosis contributes to these processes. The aim of this study was to evaluate the influence of the anti-apoptotic caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) on acute cholestatic liver injury. Methods: 8–12 week old mice exhibiting a hepatocyte-specific deletion of cFLIP using albumin driven cre-expression (Alb-Cre: FLIPf/f, cFLIP−/−) and wild type littermates were examined in a bile duct ligation (BDL) model of cholestasis. Therefore a laparotomy was performed in anesthetized mice and the common bile duct was
Journal of Hepatology 2016 vol. 64 | S631–S832
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