A proposal from the liver forum for the management of comorbidities in non-alcoholic steatohepatitis therapeutic trials.

The current document has been developed by the Liver Forum who mandated the NAFLD-Associated Comorbidities Working Group - a multistakeholder group comprised of experts from academic medicine, industry and patient associations - to identify aspects of diverse comorbidities frequently associated with non-alcoholic steatohepatitis (NASH) that can interfere with the conduct of therapeutic trials and, in particular, impact efficacy and safety results. The objective of this paper is to propose guidance for the management of relevant comorbidities in both candidates and actual participants in NASH therapeutic trials. We relied on specific guidelines from scientific societies, when available, but adapted them to the particulars of NASH trials with the aim of addressing multiple interacting requirements such as maintaining patient safety, reaching holistic therapeutic objectives, minimising confounding effects on efficacy and safety of investigational agents and allowing for trial completion. We divided the field of action into: first, analysis and stabilisation of the patient's condition before inclusion in the trial and, second, management of comorbidities during trial conduct. For the former, we discussed the concept of acceptable vs. optimal control of comorbidities, defined metabolic and ponderal stability prior to randomisation and weighed the pros and cons of a run-in period. For the latter, we analysed non-hepatological comorbid conditions for changes or acute events possibly occurring during the trial, including changes in alcohol consumption, in order to detail when specific interventions are necessary and how best to manage concomitant drug intake in line with methodological constraints. These recommendations are intended to act as a guide for clinical trialists and are open to further refinement when additional data become available.

Protocol to ensure continued pediatric liver transplantation service during the COVID pandemic and the encouraging outcomes.

Coronavirus disease 2019 is a global pandemic, and to deal with the unexpected, enormous burden on healthcare system, liver transplantation (LT) services have been suspended in many centers. Development of robust and successful protocols in preventing the disease among the recipients, donors and healthcare workers would help in re-starting the LT programs. We adapted a protocol at our center, which is predominantly a living donor liver transplant center based in north India, and continued the service as the pandemic unfolded and peaked in India with good results and shared the experience of the same. Between March 24 and June 7, 2020, during the government-enforced public curfew-"lockdown"-7 children received LT. The protocols of infection control were drafted in our team by local customization of published guidelines. The number of pediatric LT done during the lockdown period in 2020 was similar to that done in corresponding pre-COVID period in 2019. The outcomes were of 100% survival, and none of recipients developed COVID. One potential donor was asymptomatic positive for COVID, responded well to conservative treatment, and was later accepted as a donor. LT program during the COVID pandemic can successfully function after putting in place standard protocols for infection control. These can be implemented with minimal extra involvement of healthcare infrastructure, hence without diversion of resources from COVID management. In conclusion, pediatric liver transplantation services can be continued amid COVID-19 pandemic after establishing a properly observed protocol with minimum additional resources.

Outcome of Pediatric Liver Transplants in Patients With Less Than 10 kg of Body Weight Is Not Worse.

OBJECTIVES: Liver transplant in pediatric patients with body weight < 10 kg poses a challenge to the entire liver transplant team. Many reports have considered 10 kg to be a cutoff pointfor body weightforfavorable posttransplant outcomes. With evolving surgical techniques and postoperative management, there is potential to improve outcomes in this subset of recipients. We compared the outcomes in pediatric patients with body weight < 10 kg with those > 10 kg; also, we studied the factors of influence. MATERIALS AND METHODS: We performed a retrospective analysis to evaluate the outcomes of liver transplants in pediatric patients with < 10 kg body weight. The cohort consisted of 90 children subdivided into the following 2 subgroups: group A (n = 35) with > 10 kg body weight at liver transplant and group B (n = 55) with < 10 kg body weight at liver transplant. We compared the following pretransplant characteristics between the groups: graft weight, graft-to-recipient weightratio, cold ischemia time, warm ischemia times, and liver transplant outcomes. RESULTS: Pediatric End-stage Liver Disease score was significantly higher in group B (score of 24) versus group A (score of 18). Group B had significantly higher graft-to-recipient weight ratio (2.8 in group B vs 1.7 in group A). Graft function showed no significant difference between the 2 groups. Portal vein thrombosis was seen only in group B, whereas biliary leaks were observed among 5 patients in group B and 1 patientin group A. Patient survivalrate was higherin group B (86%) than in group A (77%). CONCLUSIONS: Pediatric patients weighing < 10 kg have similarif not better survivalrates after liver transplant compared with patients > 10 kg. Advancements in surgical techniques and a careful monitoring for complications and timely intervention are important to facilitate these outcomes.

Novel insights into the assessment of risk of upper gastrointestinal bleeding in decompensated cirrhotic children.

OBJECTIVES: Cirrhotic children wait-listed for liver transplant are prone to bleeding from gastrointestinal varices. Grade 2-3 esophageal varices, red signs, and gastric varices are well-known risk factors. However, the involvement of hemostatic factors remains controversial because of the rebalanced state of coagulation during cirrhosis. METHODS: Children suffering from decompensated cirrhosis were prospectively included while being on waitlist. Portal hypertension was assessed by ultrasound and endoscopy. Coagulopathy was evaluated through conventional tests, thromboelastometry, and platelet function testing. The included children were followed up until liver transplantation, and all bleeding episodes were recorded. Children with or without bleeding were compared according to clinical, radiological, endoscopic, and biological parameters. In addition, validation of a predictive model for risk of variceal bleeding comprising of grade 2-3 esophageal varices, red spots, and fibrinogen level <150 mg/dL was applied on this cohort. RESULTS: Of 20 enrolled children, 6 had upper gastrointestinal bleeding. Significant differences were observed in fibrinogen level, adenosine diphosphate, and thrombin-dependent platelet aggregation. The model used to compute the upper gastrointestinal bleeding risk had an estimated predictive performance of 81.0%. Platelet aggregation analysis addition improved the estimated predictive performance up to 89.0%. CONCLUSIONS: We demonstrated an association between hemostatic factors and the upper gastrointestinal bleeding risk. A low fibrinogen level and platelet aggregation dysfunction may predict the risk of bleeding in children with decompensated cirrhosis. A predictive model is available to assess the upper gastrointestinal bleeding risk but needs further investigations. Clinicaltrials.gov number: NCT03244332.

Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody.

This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity. The effect of a peptone/hydrolysate on coloration was investigated in cell culture experiments, which showed its use can lead to reduced coloration. Mechanisms by which these compounds influence coloration will be briefly discussed. Since it has been previously shown that antibody oxidation could potentially lead to coloration, the current effort was focused on screening for specific antioxidant additives to the culture medium to reduce coloration. An in-vitro incubation model was used to screen antioxidant compounds, several of which were found to significantly reduce antibody color, while some led to significantly increased color. Hypotaurine and carboxymethylcysteine, which had the most significant color reducing effect in the incubation study, were further tested in small-scale bioreactor cell culture experiments. These studies demonstrated that these compounds lead to reduced coloration in cell culture without affecting cell growth and titer. Hypotaurine, hydrocortisone, peptone, and cystine were also shown to reduce the acidic charge variant levels, which was previously shown to correlate with color. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1298-1307, 2018.

Living-donor liver transplantation for mild Zellweger spectrum disorder: Up to 17 years follow-up.

Mild Zellweger spectrum disorder, also described as Infantile Refsum disease, is attributable to mutations in PEX genes. Its clinical course is characterized by progressive hearing and vision loss, and neurodevelopmental regression. Supportive management is currently considered the standard of care, as no treatment has shown clinical benefits. LT was shown to correct levels of circulating toxic metabolites, partly responsible for chronic neurological impairment. Of three patients having undergone LT for mild ZSD, one died after LT, while the other two displayed significant neurodevelopmental improvement on both the long-term (17 years post-LT) and short-term (9 months post-LT) follow-up. We documented a sustained improvement of biochemical functions, with a complete normalization of plasma phytanic, pristanic, and pipecolic acid levels. This was associated with stabilization of hearing and visual functions, and improved neurodevelopmental status, which has enabled the older patient to lead a relatively autonomous lifestyle on the long term. The psychomotor acquisitions have been markedly improved as compared to their affected siblings, who did not undergo LT and exhibited a poor neurological outcome with severe disabilities. We speculate that LT performed before the onset of severe sensorineural defects in mild ZSD enables partial metabolic remission and improved long-term clinical outcomes.

Thromboelastography Parameters in Patients with Acute on Chronic Liver Failure.

INTRODUCTION AND AIM: Patients with acute on chronic liver failure (ACLF) have abnormal conventional coagulation tests- platelet count and international normalized ratio (INR). Thromboelastography (TEG) is a rapid, point-of-care assay, more comprehensive than platelet count and INR as it assesses for platelet adequacy (number and function), coagulation factors and clot retraction. The aim of the study was to evaluate the TEG parameters in patients with ACLF, chronic liver disease having acute decompensation (AD) and healthy subjects (HC). MATERIAL AND METHODS: TEG parameters were assessed in patients with ACLF and AD within 24 h of admission. Consecutive patients were included in the study over 12 months. Healthy subjects were recruited as controls. RESULTS: 179 patients were included- 68 ACLF, 53 AD and 58 HC. The mean values of INR in ACLF, AD and HC groups were 2.9 ± 1.4, 1.6 ± 0.4 and 1.1 ± 0.2; P < 0.001. Among TEG parameters - maximum amplitude (MA) was low in ACLF and AD patients as compared with HC (53.8 ± 15, 58.3 ± 13.9 mm and 67.2 ± 12.1 mm, respectively; P < 0.001). Lysis at 30 min (LY30) was high in ACLF patients, as compared to AD and HC (8.6 ± 14.1%, 5.0 ± 9.5% and 4.9 ± 9.8%, respectively; P = 0.060). There were no differences in r time, k time, and alpha angle between groups; normal in >90% patients. There was no difference in TEG parameters between different ACLF grades, whereas CCTs were more deranged with increasing grades of ACLF. CONCLUSION: Despite abnormal conventional coagulation tests, TEG parameters in ACLF patients are essentially normal, except reduced maximum amplitude. Future studies are needed to explore the utility of TEG in clinical management of ACLF patients.

Biodistribution of Liver-Derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient.

BACKGROUND: With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. METHODS: A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). RESULTS: After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. CONCLUSION: These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

The histological quantification of alpha-smooth muscle actin predicts future graft fibrosis in pediatric liver transplant recipients.

Activated hepatic stellate cells express cytoplasmic ASMA prior to secreting collagen and consequent liver fibrosis. We hypothesized that quantifying ASMA could predict severity of future fibrosis after LT. For this, 32 pairs of protocol biopsies, that is, "baseline" and "follow-up" biopsies taken at 1- to 2-year intervals from 18 stable pediatric LT recipients, transplanted between 2006 and 2012 were selected. Morphometric quantification of "ASMA-positive area percentage" was performed on the baseline biopsy. Histological and fibrosis assessment using Metavir and LAFSc was performed on all biopsies. The difference of fibrosis severity between the "baseline" and "follow-up" was termed "prospective change in fibrosis." Significant association was seen between extent of ASMA positivity on baseline biopsy and "prospective change in fibrosis" using Metavir (P=.02), cumulative LAFSc (P=.02), and portal LAFSc (P=.01) values. ASMA-positive area percentage >1.05 predicted increased fibrosis on next biopsy with 90.0% specificity. Additionally, an association was observed between extent of ASMA positivity and concomitant ductular reaction (P=.06), but not with histological inflammation in the portal tract or lobular area. Hence, ASMA quantification can predict the future course of fibrosis.

Retargeting of bile salt export pump and favorable outcome in children with progressive familial intrahepatic cholestasis type 2.

UNLABELLED: We investigated predictors of clinical evolution in progressive familial intrahepatic cholestasis type 2 patients and how they relate to bile salt export pump (BSEP) expression and its (re)targeting. Our retrospective study included 22 children with progressive familial intrahepatic cholestasis type 2. Clinical, biochemical, and histological characteristics were reviewed on admittance and following treatment with either ursodeoxycholic acid alone (10 mg/kg thrice daily, n = 19) or partial biliary diversion (n = 3). Immunostaining of BSEP was performed in 20 patients. Response to treatment was defined as normalization of pruritus, disappearance of jaundice, and alanine aminotransferase (ALT) levels <1.5 times the upper limit of normal. Ten of 22 patients were responders, and paired biopsies were available in six. De novo or retargeted canalicular expression of BSEP occurred in four of these six, two of whom exhibited baseline intracellular expression. Twelve of 22 were nonresponders and exhibited earlier onset of jaundice (<9 months), neonatal cholestasis, and higher ALT levels. An ALT >165 IU/L produced 72% sensitivity and 55% specificity in predicting nonresponse. Seven patients were still responding at last follow-up (median = 20 months, range 5-67 months). Three responders relapsed after 56, 72, and 82 months, respectively. Of nine surviving responders, median relapse-free survival time was 72 months (95% confidence interval 48-96 months) and 5-year relapse-free survival was 75% (95% confidence interval 33-100%). Intracellular BSEP at baseline was seen in six, of whom five were responders. Genetic analysis was performed in 17 of 22, confirming diagnosis in 13 (76%) and in four (24%) in whom only heterozygous mutation was identified. CONCLUSION: De novo or retargeted canalicular expression of BSEP occurs in treatment responders; children with late-onset presentation, lower ALT, and intracellular BSEP expression are likely to respond, at least transiently, to nontransplant treatment.

Effect of cell culture medium components on color of formulated monoclonal antibody drug substance.

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B-vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed.

Microscopic colitis in children with chronic diarrhea.

OBJECTIVE: The aim of the present study was to study microscopic colitis (MC) in children with special reference to its role in chronic diarrhea and changes in mucosal biopsies. METHODS: A total of 100 consecutive children ages 3 to 12 years, with nonbloody diarrhea (passage of ≥3 loose stools per day) of >12 weeks' duration were screened and 26 were enrolled in the study in which no specific etiology could be found and colonoscopy did not reveal any mucosal abnormality. Colonic biopsies were evaluated for the presence of lymphocytic colitis or collagenous colitis and those with the characteristic changes were defined to have MC (group A). Colonic biopsies from patients with MC were compared with biopsies from patients with chronic diarrhea but no evidence of MC (group B). One hundred children ages 3 to 12 years with bleeding per rectum were screened and colonic biopsies from 45 patients (group C) who had colonic mucosal changes but no vascular or polyp lesion were compared with patients with MC. RESULTS: Of the 26 patients with chronic diarrhea, MC was found in 5 (3 lymphocytic colitis and 2 collagenous colitis). Significantly higher polymorphonuclear infiltration was seen in group A as compared with group B (13.8 [5.4-20.6] vs 7.2 [0-19.6]; P = 0.03) or group C (13.8 [5.4-20.6] vs 4 [0-13.4]; P = 0.007). Intraepithelial lymphocytes (12 [4-32] vs 4 [0-24]; P = 0.008) and basement membrane thickening (3.5 [2.9-10.6] vs 2.5 [1.6-5.86]; P = 0.008) were also significantly higher in group A as compared with group C. CONCLUSIONS: MC was found to be present in children with nonbloody chronic diarrhea in children. Further multicentric studies may provide adequate data on its prevalence.