Functional Characterization of Organoids Derived From Irreversibly Damaged Liver of Patients With NASH.

BACKGROUND AND AIMS: NASH will soon become the leading cause of liver transplantation in the United States and is also associated with increased COVID-19 mortality. Currently, there are no Food and Drug Administration-approved drugs available that slow NASH progression or address NASH liver involvement in COVID-19. Because animal models cannot fully recapitulate human NASH, we hypothesized that stem cells isolated directly from end-stage liver from patients with NASH may address current knowledge gaps in human NASH pathology. APPROACH AND RESULTS: We devised methods that allow the derivation, proliferation, hepatic differentiation, and extensive characterization of bipotent ductal organoids from irreversibly damaged liver from patients with NASH. The transcriptomes of organoids derived from NASH liver, but not healthy liver, show significant up-regulation of proinflammatory and cytochrome p450-related pathways, as well as of known liver fibrosis and tumor markers, with the degree of up-regulation being patient-specific. Functionally, NASH liver organoids exhibit reduced passaging/growth capacity and hallmarks of NASH liver, including decreased albumin production, increased free fatty acid-induced lipid accumulation, increased sensitivity to apoptotic stimuli, and increased cytochrome P450 metabolism. After hepatic differentiation, NASH liver organoids exhibit reduced ability to dedifferentiate back to the biliary state, consistent with the known reduced regenerative ability of NASH livers. Intriguingly, NASH liver organoids also show strongly increased permissiveness to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vesicular stomatitis pseudovirus as well as up-regulation of ubiquitin D, a known inhibitor of the antiviral interferon host response. CONCLUSION: Expansion of primary liver stem cells/organoids derived directly from irreversibly damaged liver from patients with NASH opens up experimental avenues for personalized disease modeling and drug development that has the potential to slow human NASH progression and to counteract NASH-related SARS-CoV-2 effects.

Liver Transplantation for Hepatic Epithelioid Hemangioendothelioma Is Facilitated by Exception Points With Acceptable Long-term Outcomes.

BACKGROUND: Hepatic epithelioid hemangioendothelioma (HEHE) is a rare vascular tumor with a high mortality rate. HEHE is now a formally recognized indication for exception point priority in the United States under the new National Liver Review Board. The role of liver transplantation (LT) and exception point waitlist priority in the United States for patients with HEHE remains understudied. METHODS: This was a retrospective cohort study using the United Network for Organ Sharing transplant database. From February 27, 2002 to January 31, 2018, 131 adults waitlisted for LT with HEHE were identified by free-text entry. RESULTS: Exception point applications were submitted for 91.6% (120/131) of patients. All patients with fully reviewed applications received exception points at least once during waitlisting, and 85% (103/120) upon first submission. Among the 88 patients transplanted, median model for end-stage liver disease score at LT was 7 ((interquartile range [IQR]: 6-11) and waiting time 78.5 days (IQR: 29.5-237.5). Unadjusted post-LT survival of HEHE recipients at 1-, 3-, and 5-years from LT was 88.6%, 78.9%, and 77.2%. Unadjusted post-LT patient and graft survival of HEHE patients was not different from patients with hepatocellular carcinoma within Milan receiving exception point priority (P = 0.08). An increased rate of graft failure due to hepatic artery thrombosis ≤14 days from initial LT was observed in HEHE versus non-HEHE patients (4.6% versus 0.5%). CONCLUSIONS: The majority of HEHE recipients receive exception points at a universal approval rate allowing prompt access to deceased donor LT.

N-Acetyl Cysteine Attenuates the Sarcopenia and Muscle Apoptosis Induced by Chronic Liver Disease.

BACKGROUND: Sarcopenia is characterized by the loss of muscle mass and strength (muscle atrophy) because of aging or chronic diseases, such as chronic liver disease (CLD). Different mechanisms are involved in skeletal muscle atrophy, including decreased muscle fibre diameter and myosin heavy chain levels and increased ubiquitin-proteasome pathway activity, oxidative stress and myonuclear apoptosis. We recently found that all these mechanisms, except myonuclear apoptosis, which was not evaluated in the previous study, were involved in muscle atrophy associated with hepatotoxin 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced CLD. OBJECTIVE: In the present study, we evaluated the involvement of myonuclear apoptosis in CLD-associated sarcopenia and the effect of N-acetyl cysteine (NAC) treatment on muscle strength and apoptosis, using a DDC-supplemented diet-fed mouse model. METHODS: Four-month-old male C57BL6 mice were fed with a standard or DDCsupplemented diet for six weeks in the absence or presence of NAC treatment. RESULTS: Our results showed that NAC attenuated the decrease in muscle fibre diameter and muscle strength associated with CLD-induced muscle wasting in gastrocnemius (GA) muscle of DDC-supplemented diet-fed mice. In addition, in GA muscle of the mice fed with DDC-supplemented diet-induced CLD showed increased myonuclear apoptosis compared with the GA muscle of the control diet-fed mice, as evidenced by increased apoptotic nuclei number, caspase-8 and caspase-9 expression, enzymatic activity of caspase-3 and BAX/BCL-2 ratio. NAC treatment inhibited all the mechanisms associated with myonuclear apoptosis in the GA muscle. CONCLUSION: To our knowledge, this is the first study which reports the redox regulation of muscle strength and myonuclear apoptosis in CLD-induced sarcopenia.

Diuretic window hypothesis in cirrhosis: Changing the point of view.

Since the 1970s, non-selective beta-blockers (NSBB) have been used to prevent variceal upper bleeding in advanced cirrhotic patients. However, several recent studies have raised the doubt about the benefit of NSBB in end-stage cirrhotic patients. In fact, they suggested a detrimental effect in these patients that even reduced survival. All of these studies have been assembled to compose the "window therapy hypothesis", in which NSBB would have traditional indication to be initiated to prevent variceal upper bleeding; however, treatment should be stopped (or not be initiated) in patients with end-stage cirrhosis. NSBB would reduce the cardiac reserve of these patients, worsening systemic perfusion and prognosis. However, it should be emphasized that these studies present important bias issues, and their results also suggested that diuretic treatment may also be behind the effects observed. In this opinion review, we changed the point of view from NSBB to diuretic treatment, based on a physiopathogenic approach of circulatory parameters of cirrhotic patients studied, and based on diuretic effect in blood pressure lowering and in other hypervolemic disease, as heart failure. We suggest a "diuretic window hypothesis", composed by an open window in hypervolemic phase, an attention window when patient present in a normal plasma volume phase, and a closed window during the plasma hypovolemic phase.

Candesartan, rather than losartan, improves motor dysfunction in thioacetamide-induced chronic liver failure in rats.

Minimal hepatic encephalopathy is more common than the acute syndrome. Losartan, the first angiotensin-II receptor blocker (ARB), and candesartan, another widely-used ARB, have protected against developing fibrogenesis, but there is no clear data about their curative antifibrotic effects. The current study was designed to examine their effects in an already-established model of hepatic fibrosis and also their effects on the associated motor dysfunction. Low-grade chronic liver failure (CLF) was induced in 3-month old Sprague-Dawley male rats using thioacetamide (TAA, 50 mg·kg-1·day-1) intraperitoneally for 2 weeks. The TAA-CLF rats were randomly divided into five groups (n=8) treated orally for 14 days (mg·kg-1·day-1) as follows: TAA (distilled water), losartan (5 and 10 mg/kg), and candesartan (0.1 and 0.3 mg/kg). Rats were tested for rotarod and open-field tests. Serum and hepatic biochemical markers, and hepatic histopathological changes were evaluated by H&E and Masson's staining. The TAA-CLF rats showed significant increases of hepatic malondialdehyde, hepatic expression of tumor necrosis factor-α (TNF-α), and serum ammonia, alanine aminotransferase, γ-glutamyl transferase, TNF-α, and malondialdehyde levels as well as significant decreases of hepatic and serum glutathione levels. All treatments significantly reversed these changes. The histopathological changes were moderate in losartan-5 and candesartan-0.1 groups and mild in losartan-10 and candesartan-0.3 groups. Only candesartan significantly improved TAA-induced motor dysfunction. In conclusion, therapeutic antifibrotic effects of losartan and candesartan in thioacetamide-induced hepatic fibrosis in rats are possibly through angiotensin-II receptor blocking, antioxidant, and anti-inflammatory activities. Improved motor dysfunction by candesartan could be attributed to better brain penetration and slower "off-rate" from angiotensin-II receptors. Clinical trials are recommended.

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia.

BACKGROUND: Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. METHODS: Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. RESULTS: In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. CONCLUSIONS: This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis.

Candesartan, rather than losartan, improves motor dysfunction in thioacetamide-induced chronic liver failure in rats

Minimal hepatic encephalopathy is more common than the acute syndrome. Losartan, the first angiotensin-II receptor blocker (ARB), and candesartan, another widely-used ARB, have protected against developing fibrogenesis, but there is no clear data about their curative antifibrotic effects. The current study was designed to examine their effects in an already-established model of hepatic fibrosis and also their effects on the associated motor dysfunction. Low-grade chronic liver failure (CLF) was induced in 3-month old Sprague-Dawley male rats using thioacetamide (TAA, 50 mg·kg−1·day−1) intraperitoneally for 2 weeks. The TAA-CLF rats were randomly divided into five groups (n=8) treated orally for 14 days (mg·kg−1·day−1) as follows: TAA (distilled water), losartan (5 and 10 mg/kg), and candesartan (0.1 and 0.3 mg/kg). Rats were tested for rotarod and open-field tests. Serum and hepatic biochemical markers, and hepatic histopathological changes were evaluated by H&E and Masson's staining. The TAA-CLF rats showed significant increases of hepatic malondialdehyde, hepatic expression of tumor necrosis factor-α (TNF-α), and serum ammonia, alanine aminotransferase, γ-glutamyl transferase, TNF-α, and malondialdehyde levels as well as significant decreases of hepatic and serum glutathione levels. All treatments significantly reversed these changes. The histopathological changes were moderate in losartan-5 and candesartan-0.1 groups and mild in losartan-10 and candesartan-0.3 groups. Only candesartan significantly improved TAA-induced motor dysfunction. In conclusion, therapeutic antifibrotic effects of losartan and candesartan in thioacetamide-induced hepatic fibrosis in rats are possibly through angiotensin-II receptor blocking, antioxidant, and anti-inflammatory activities. Improved motor dysfunction by candesartan could be attributed to better brain penetration and slower "off-rate" from angiotensin-II receptors. Clinical trials are recommended.

Relationships between transient elastography values and liver fibrosis in chronic liver disease patients with normal or mildly abnormal aminotransferase levels.

This study aimed to evaluate relationships between transient elastography values and liver fibrosis in chronic liver disease patients with normal or mildly abnormal aminotransferase levels. Fifty-six patients were enrolled in the study. Transient elastography and liver biopsy were performed on the same day, and the fibrosis was staged based on the Scheuer scoring system. Liver stiffness was measured to assessed liver fibrosis using transient elastography. The transient elastography values of 12 patients with chronic hepatitis B were studied before and 6 months after antiviral treatment. The sensitivity and specificity for 10.88 kPa in S3 were 80 and 87.8%, and for 19.4 kPa in S4, were 100 and 90.7%, respectively. In univariate analysis, liver stiffness strongly correlated with the fibrosis stage (r = 0.70, P < 0.5), moderately correlated with the aminotransferases (r = 0.398, P < 0.05), and poorly correlated with the degree of necroinflammatory activity (r = 0.19, P < 0.5). In multivariate regression, liver stiffness correlated only with the fibrosis stage (P < 0.05). Pre- and post-treatment viral loads were not significantly different [(4.81 ± 0.15) x 10(6) vs (7.62 ± 0. 16) x 10(3), P > 0.05]. Pre- and post-treatment LS measurements were not correlated with viral load (P > 0.05). Pre- and post-treatment LS measurements were not significantly different (P > 0.02). In conclusion, transient elastography values correlated with the stage of cirrhosis, alanine aminotransferase levels, and antiviral treatment in patients with chronic hepatitis B and did not correlate with viral loads.

Role of MELD score and serum creatinine as prognostic tools for the development of acute kidney injury after liver transplantation.

BACKGROUND: The role of the Model for End-Stage Liver Disease (MELD) score in predicting complications, such as Acute Kidney Injury (AKI), after orthotopic liver transplantation (OLT) has yet to be evaluated and serum creatinine may be too heavily weighted in the existing MELD formula, since it has many pitfalls in cirrhotic patients. METHODS: Retrospective data of the perioperative period from consecutive adult OLTs performed from January to December 2009 were recorded. Univariate and multivariate analysis were performed to analyze the risk factors for AKI and mortality after OLT. RESULTS: There were 114 OLTs performed in the study period, 22 (19,2%) were submitted to dialysis prior OLT and were excluded from the analysis for AKI. The median age was 52 years and 66% were male. Median creatinine value was 0.85mg/dL and MELD was 19. Fifty-two of the 92 patients (56,5%) developed AKI in the first 72 hours after OLT. The only independent risk factor for AKI was calculated MELD and when the components of the MELD score were analyzed, INR had a much stronger impact in predicting AKI then serum creatinine. Overall mortality rate was 32,5% and anesthesia duration was the only variable associated with higher mortality rate. CONCLUSIONS: Although MELD score seems to have a good performance in predicting AKI after OLT, serum creatinine had no impact on its prediction despite its importance on MELD calculation. Modifying the MELD score, which could include novel AKI biomarkers, may improve its prognostic accuracy and provide a better tool for public health planning.

Impact of Model for End-Stage Liver Disease in the occurrence of infectious events and survival in a cohort of liver transplant recipients.

The Model for End-Stage Liver Disease (MELD), which predicts mortality on the waiting list before liver transplantation, has changed organ allocation criteria to prioritize severely ill patients. The aim of this study was to investigate the impact of the new criteria on the incidence of Healthcare Associated Infections (HAI) and patient survival after liver transplantation. This retrospective cohort included liver transplant recipients from 2005 to 2007. Infection notification followed the recommended criteria of the National Healthcare Safety Network (NHSN). Statistical analysis was performed using the Statistical Package for the Social Sciences. Of 142 patients, 67 (47.2%) underwent transplantation before June 2006. There were no differences between the 2 periods considering patient gender, diagnosis, age, length of hospitalization, and mean time to first infection occurrence. However, the length of intensive care unit (ICU) hospitalization (P = .006) and central venous catheter (CVC) use (P = .025) were higher in the first period of the study. Comparison of time until first systemic infection before and after changes in allocation criteria showed no significant difference (log-rank = 0.06; P = .81). There was a trend toward greater lethality during the second period of the study (P = .09). There was no difference in time to death between the 2 periods (log-rank = 0.9; P = .76). However, when comparing time to death of all patients with systemic infection versus those without this event, patients without infection showed a higher mortality rate (log-rank = 15.7; P < .001).

Impact of the severity of end-stage liver disease in cardiac structure and function.

BACKGROUND: The impact of end-stage liver disease (ESLD) in cardiac remodeling of patients with cirrhosis is unknown. Our aim was to correlate the severity of ESLD with morphologic and functional heart changes. MATERIAL AND METHODS: 184 patients underwent a protocol providing data on the severity of ESLD and undergoing echocardiography to assess the diameters of the left atrium and right ventricle; the systolic and diastolic diameters of the left ventricle, interventricular septum, and posterior wall of the left ventricle; systolic pulmonary artery pressure; ejection fraction; and diastolic function. Severity of ESLD was assessed by the Model for End-Stage Liver Disease (MELD) score. RESULTS: Left-atrial diameter (r = 0.323; IC 95% 0.190-0.455; p < 0.001), left-ventricular diastolic diameter (r = 0.177; IC 95% 0.033-0.320; p = 0.01) and systolic pulmonary artery pressure (r = 0.185; IC 95% 0.036-0.335; p = 0.02) significantly correlated with MELD score. Patients with MELD ≥ 16 had significantly higher left-atrial diameter and systolic pulmonary artery pressure, compared with patients with MELD scores < 16 points. CONCLUSIONS: Changes in cardiac structure and function correlate with the severity of ESLD.

Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats.

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior.