Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

AbstractBackground aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 104 to 28.3 × 104 cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: diagnostic and mechanistic relevance

Background & Aims: Serum microRNAs (miRNAs) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages.Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 NAFLD cases representing the complete NAFLD spectrum and 10 population controls). MiRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional NAFLD cases and 15 population controls by quantitative reverse transcriptase-polymerase chain reaction.Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages but miR-193a-5p consistently the showed increased levels in all comparisons. Relative to NAFL/NASH with mild fibrosis (stage 0/1), three miRNAs (miR-193a-5p, miR-378d and miR378d) were increased in cases with NASH and clinically significant fibrosis (stage 2-4), seven (miR193a-5p, miR-378d, miR-378e, miR-320b, c, d & e) increased in cases with NAFLD Activity Score (NAS) 5-8 compared with lower NAS, and three (miR-193a-5p, miR-378d, miR-378e) increased but one (miR-19b-3p) decreased in steatosis, activity, and fibrosis "activity" (SAF-A) score 2-4 compared with lower SAF-A. The significant findings for miR-193a-5p were replicated in the additional NAFLD cohort. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n=80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD

The search for disease-modifying agents in decompensated cirrhosis: From drug repurposing to drug discovery.

Patients with decompensated cirrhosis are currently managed through targeted strategies aimed at preventing or treating specific complications. In contrast, a disease-modifying agent should, by definition, be aimed at globally addressing 'decompensated cirrhosis'. To be defined as a disease-modifying agent in decompensated cirrhosis, interventions need to demonstrate an unequivocal benefit on the course of disease in well-designed and adequately powered randomised clinical trials with hard endpoints (i.e. patient survival). These trials also need to define the target population, dosage and timing of administration, factors guiding treatment, temporary or permanent stopping rules, transferability to daily clinical practice, cost-effectiveness, and global treatment access. By eliminating the underlying cause of cirrhosis, aetiologic treatments can still influence the course of decompensated disease by halting or slowing down disease progression or even inducing reversion to the compensated state. In contrast, there remains an unmet clinical need for disease-modifying agents which can antagonise key pathophysiological mechanisms of decompensated cirrhosis, such as portal hypertension, gut translocation, circulatory dysfunction, systemic inflammation, and immunological dysfunction. However, in the last few years, the repurposing of "old drugs" that have already been prescribed for more limited indications in hepatology or for other diseases has provided a few candidates, including human albumin, statins, and poorly absorbable oral antibiotics, which are under further evaluation in large-scale randomised clinical trials. New disease-modifying agents are also expected to be identified in the next decade through the systematic repurposing of existing drugs and the development of novel molecules which are currently undergoing pre-clinical or early clinical testing

Prevalence of High-risk Non-alcoholic Steatohepatitis (NASH) in the United States: Results from NHANES 2017-2018.

BACKGROUND & AIMS The population prevalence of high-risk non-alcoholic steatohepatitis (NASH), defined as NAFLD activity score ≥4 and fibrosis stage ≥2, is unknown. FAST score, calculated using LSM and CAP values from FibroScan® and AST levels, is a validated algorithm to identify individuals with high-risk NASH. We estimated the prevalence of high-risk NASH using the FAST score in the U.S. POPULATION METHODS Data were derived from the National Health and Nutrition Examination Surveys 2017-2018, which included a total of 4218 adults with valid elastography measurements. FAST scores of ≥0.35 (sensitivity 90%) and ≥0.67 (specificity 90%) were used to identify adults with high-risk NASH in the general population. RESULTS At 90% sensitivity for the FAST score, the prevalence of age-adjusted high-risk NASH was 5.8% and was higher among men (8.2% vs. 3.6% in women) and in Hispanics (9.2% vs. 5.8% non-Hispanic (N.H.) Asians, 5.2% in N.H. Whites, and 3.8% in N.H. Blacks). The prevalence of high-risk NASH was 11.7% in those with metabolic syndrome (MetS) and 22.5% in individuals with type 2 diabetes mellitus (T2DM). At 90% specificity for the FAST score, the prevalence of age-adjusted high-risk NASH was 1.2% and was higher among men (1.7% vs. 0.8% in women) and in Hispanics (2.2% vs. 1.0% in N.H. Asians, 0.9% in N.H. Whites, and 0.4% in N.H. Blacks). The prevalence of high-risk NASH was 3.4% in those with MetS and 8.7% in adults with T2DM. CONCLUSIONS We estimate at least 2 million adults have high-risk NASH in the US. Moreover, the prevalence of high-risk NASH among individuals with T2DM is higher ranging between 8.7% and 22.5%, supporting the case for coordinated case-finding and management

Annual Brome Biocontrol after Wildfire Using a Native Fungal Seed Pathogen

A major problem in post-fire restoration of semi-arid shrublands dominated by annual bromes is the presence of carryover seed banks that cannot be controlled using conventional methods. These seeds can provide significant competition for seeded species in the years following treatment. We investigated the feasibility of using a naturally occurring seed pathogen, the ascomycete Pyrenophora semeniperda, as a biocontrol organism for eliminating this carryover seed bank. We carried out the necessary technology development to create and apply field inoculum to cheatgrass- or red brome-infested areas (both burned and unburned) at six sites located in three states across two years of field trials. We found that inoculum application significantly increased the proportion of pathogen-killed Bromus seeds in the seed bank, reduced the density of viable carryover Bromus seeds, and in many cases increased the density of pathogen-killed seeds relative to levels in uninoculated controls. In some treatments, the proportion of field-killed seeds reached 100%, validating the promise of this approach. Even though this pathogen is physiologically capable of infecting the seeds of many grasses and some dicots, we determined that risks to nontarget host seeds can be mitigated. The inoculum usually has a relatively short persistence time in the absence of new host seeds, and the pathogen is readily controlled by fungicides that could potentially be used as seed treatments for desired restoration species. The potential for selection of more virulent P. semeniperda strains for increased biocontrol effectiveness is considerable. In addition, because more virulent strains grow more slowly, they are less likely to persist post-control in competition with faster-growing wild strains. In summary, our study provides proof of concept for use of this pathogen for biocontrol of cheatgrass and red brome, and opens the way for further studies on formulation and delivery technology to bring this promising biocontrol agent closer to market

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

Background aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 104 to 28.3 × 104 cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research