Kevin Moore

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models

Background Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. Methods and findings In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. Conclusions The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.

Characterisation of Agomelatine-Induced Increase in Liver Enzymes: Frequency and Risk Factors Determined from a Pooled Analysis of 7605 Treated Patients

BackgroundAntidepressant-induced liver injury is a major concern and a liver monitoring scheme has been recommended by the European Medicines Agency for agomelatine.ObjectiveThe objective of this study was to assess the liver safety and identify the characteristics of patients who developed a significant increase in transaminases whilst taking agomelatine.MethodA retrospective pooled analysis of changes in transaminase levels in 9234 patients treated with agomelatine (25 or 50xa0mg/day; nxa0=xa07605) or placebo (nxa0=xa01629) from 49 phasexa0II and III studies was undertaken. A significant increase in transaminase levels was defined as an increase to >3 times the upper limit of normal (ULN) (>3xa0×xa0ULN). Final causality was determined in a case-by-case review by five academic experts.ResultsSerum transaminases increased to >3xa0×xa0ULN in 1.3 and 2.5xa0% of patients treated with 25 and 50xa0mg of agomelatine, respectively, compared with 0.5xa0% for placebo. The onset of increased transaminases occurred before 12xa0weeks in 64xa0% of patients. The median time to recovery (to ≤2xa0×xa0ULN) was 14xa0days following treatment withdrawal. Liver function tests recovered in 36.1xa0% of patients despite continuation of agomelatine, suggesting the presence of a liver adaptive mechanism. No cases of acute liver failure or fatal outcome occurred. Patients with elevated transaminases at baseline, secondary to obesity/fatty liver disease, had an equally increased risk of developing further elevations of transaminases with agomelatine and placebo.ConclusionIncidence of abnormal transaminases was low and dose dependent. No specific population was identified regarding potential risk factors. Withdrawal of agomelatine led to rapid recovery, and some patients exhibited an adaptive phenomenon. Overall, in clinical trials, the liver profile of agomelatine seems safe when serum transaminases are monitored.

DIAMOND (DIgital Alcohol Management ON Demand): a mixed methods feasibility RCT and embedded process evaluation of a digital health intervention to reduce hazardous and harmful alcohol use

BackgroundAlcohol is a major risk factor for preventable illness, with huge cost to healthcare economies. There is a role for alcohol-specific digital health interventions (DHI), but there have been few randomised controlled trials (RCT) comparing DHI with face-to-face treatment. Such trials are complex and face obstacles in recruitment and retention.MethodsMixed-methods feasibility RCT of an alcohol DHI, testing recruitment, online data-collection and randomisation processes, with an embedded process evaluation. Recruitment ran from October 2014 for 9xa0months. Participants were adults drinking at hazardous and harmful levels, attending four community drug and alcohol services (CDAS) in London. Participants completed baseline demographic, alcohol-related and other psychological questionnaires online and were randomised to HeLP-Alcohol, a six-module DHI with weekly reminder prompts (phone, email or text message), which mirrors face-to-face treatment, or to face-to-face treatment at CDAS. Alcohol counsellors took part in qualitative interviews at the end of the study.ResultsAlcohol counsellors screened 1253 patients. One thousand one hundred eighty-nine did not meet inclusion criteria so were excluded: 579 were dependent drinkers, 548 had health conditions that made them ineligible to take part and 62 were ineligible for other reasons including homelessness. Of the 64 patients who were eligible to take part, 54 declined to participate, with 36 stating a preference for face-to-face treatment, 13 gave no reason, and 5 gave other reasons including not wanting to use a computer. Ten consented but then 3 changed their minds, so we were able to randomise 7 participants to the study (11% of eligible).Five alcohol counsellors agreed to be interviewed for the process evaluation and provided the following feedback: Although most of their colleagues were enthusiastic about the trial, some were not at equipoise in recruiting; potential participants also declared strong preference to intervention arm from the outset. These factors affected recruitment. Counsellors also lacked time to undertake the data inputting and follow-up of participants in addition to their everyday work.ConclusionsThis feasibility study aimed to test recruitment, randomisation, retention and data collection methods but recruited only 7 participants so these aims were not fully achieved. This illustrates to all researchers of complex interventions the importance of conducting feasibility studies and is generalisable to areas other than alcohol research.CDAS were seeing larger numbers of non-dependent drinkers with complex additional problems than alcohol commissioners expected. CDAS clients and some counsellors were not at equipoise for recruitment. Alternative settings for recruitment need to be explored in future trials.Trial registrationInternational Standard Randomized Controlled Trial Number: ISRCTN31789096, DOI 10.1186/ISRCTN31789096

Development of human liver extracellular matrix hydrogel for three dimensional cell culture and cell transplantation

Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications.

Short-term abstinence from alcohol and changes in cardiovascular risk factors, liver function tests and cancer-related growth factors: a prospective observational study

Objective To assess changes in metabolic risk factors and cancer-related growth factors associated with short-term abstinence from alcohol. Design Prospective, observational study. Setting Single tertiary centre. Participants Healthy subjects were recruited based on intention to: (1) abstain from alcohol for 1u2009month (abstinence group), or (2) continue to drink alcohol (control group). Inclusion criteria were baseline alcohol consumption >64u2009g/week (men) or >48u2009g/week (women). Exclusion criteria were known liver disease or alcohol dependence. Primary and secondary outcome measures The primary outcome was change in insulin resistance (homeostatic model assessment (HOMA) score). Secondary outcomes were changes in weight, blood pressure (BP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and liver function tests. Primary and secondary outcomes were adjusted for changes in diet, exercise and cigarette smoking. Results The abstinence group comprised 94 participants (mean age 45.5 years, SD ±1.2) and the control group 47 participants (mean age 48.7 years, SD ±1.8). Baseline alcohol consumption in the abstinence group was 258.2u2009g/week, SD ±9.4, and in the control group 233.8u2009g, SD ±19.0. Significant reductions from baseline in the abstinence group (all p<0.001) were found in: HOMA score (−25.9%, IQR −48.6% to +0.3%), systolic BP (−6.6%, IQR −11.8% to 0.0%), diastolic BP (−6.3%, IQR −14.1% to +1.3%), weight (−1.5%, IQR −2.9% to −0.4%), VEGF (−41.8%, IQR −64.9% to −17.9%) and EGF (−73.9%, IQR −86.1% to −36.4%). None of these changes were associated with changes in diet, exercise or cigarette smoking. No significant changes from baseline in primary or secondary outcomes were noted in the control group. Conclusion These findings demonstrate that abstinence from alcohol in moderate–heavy drinkers improves insulin resistance, weight, BP and cancer-related growth factors. These data support an independent association of alcohol consumption with cancer risk, and suggest an increased risk of metabolic diseases such as type 2 diabetes and fatty liver disease.

Acute hepatitis E mimicking a flare of disease in a patient with chronic autoimmune hepatitis.

xa0Acute hepatitis E is becoming increasingly recognised in Europe with up to 40% of the population in Southern France being exposed to the virus, which is harboured in pigs. Patients with known liver disease may present with acute hepatitis E and present a diagnostic challenge. For example patients with autoimmune hepatitis (AIH) who are immunosuppressed and contract hepatitis E may be at increased risk of developing chronicity due to concurrent immunosuppression. Importantly, the diagnosis may be missed with the infection misdiagnosed as an autoimmune flare, and immunosuppression increased by the attending physician, thus enhancing the risk of chronicity of infection leading to progressive liver injury in immunocompromised patients. We report a case of acute hepatitis E in a patient with AIH and discuss the features that helped us differentiating it from an autoimmune flare.Acute hepatitis E is becoming increasingly recognised in Europe with up to 40% of the population in Southern France being exposed to the virus, which is harboured in pigs. Patients with known liver disease may present with acute hepatitis E and present a diagnostic challenge. For example patients with autoimmune hepatitis (AIH) who are immunosuppressed and contract hepatitis E may be at increased risk of developing chronicity due to concurrent immunosuppression. Importantly, the diagnosis may be missed with the infection misdiagnosed as an autoimmune flare, and immunosuppression increased by the attending physician, thus enhancing the risk of chronicity of infection leading to progressive liver injury in immunocompromised patients. We report a case of acute hepatitis E in a patient with AIH and discuss the features that helped us differentiating it from an autoimmune flare.