Marianne Samyn

Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis

CD4+CD25+ regulatory T cells (T‐regs) are central to the maintenance of immune tolerance and represent an immune intervention candidate in autoimmune hepatitis (AIH), a condition characterized by impaired T‐reg number and function. We investigated whether T‐regs can be expanded from the existing CD4+CD25+ T cell pool and generated de novo from CD4+CD25− T cells in AIH patients and healthy controls. Purified CD4+CD25+ and CD4+CD25− T cells from 24 patients with type 1 AIH and 22 healthy controls were cultured for up to 5 weeks with anti‐CD3/anti‐CD28 T cell expander and high‐dose interleukin‐2 (IL‐2). Cell phenotypes, suppressor ability, forkhead winged/helix transcription factor box P3 (FOXP3) gene, and protein expression were assessed weekly by cytofluorimetry, proliferation assay, real‐time polymerase chain reaction (PCR), and immunoblot. During culture, the number of CD4+CD25+ T cells derived from the existing T‐reg pool (expanded T‐regs) and generated de novo from CD4+CD25− T cells (newly generated T‐regs) increased constantly up to week 4 in both healthy controls and, to a lesser extent, in AIH patients. Expanded T‐regs retained conventional T‐reg phenotype and, compared with baseline, demonstrated more vigorous suppressive function and increased FOXP3 gene and protein expression. Newly generated T‐regs not only acquired T‐reg phenotype but underwent greater growth and were more resistant to apoptosis than expanded T‐regs. Their suppressive function augmented throughout culture, reaching a peak at week 4, preceded by a peak FOXP3 gene and protein expression at week 2. Suppressor function and FOXP3 expression of both expanded and newly generated T‐regs were higher in normal controls than in AIH patients. Conclusion: Functionally enhanced T‐regs can be expanded and generated de novo in patients with AIH. This finding may assist in reconstituting impaired immune regulation and restoring peripheral tolerance through T‐reg infusion in this condition. (HEPATOLOGY 2008;47:581–591.)

Mycophenolate mofetil as rescue treatment for autoimmune liver disease in children: A 5-year follow-up

BACKGROUND/AIM The aim of this study was to evaluate the outcome of mycophenolate mofetil (MMF) therapy in children with autoimmune liver disease who are resistant to or intolerant of standard immunosuppression. METHODS INCLUSION CRITERIA (a) failure to achieve/maintain remission with prednisolone/azathioprine therapy or (b) significant treatment side-effects. Initial MMF dose was 20mg/kg/day, gradually increased to a maximum of 40 mg/kg/day. Azathioprine was stopped when MMF was commenced. RESULTS Twenty-six children (17 female) were recruited. Median (range) age at diagnosis was 9.9 (1.2-14.4) years. Sixteen had Type 1 autoimmune hepatitis (AIH), two Type 2 AIH, and eight had autoimmune sclerosing cholangitis (ASC). Median (range) time from diagnosis to addition of MMF was 14.9 (0.2-108.6) months. Eighteen children responded to MMF, aspartate aminotransferase (AST) normalising in 14. At median (range) follow-up of 61.5 (19.5-96.3) months, AST remained normal in 12. All 18 children were well, but two had clinical signs of portal hypertension. Eight (6 ASC) did not respond: AST remained elevated in seven, one was listed for transplant for decompensated liver disease and one had clinical signs of portal hypertension. MMF was well tolerated. Leukopenia (n=7) was the most common side-effect. CONCLUSIONS MMF is an effective rescue therapy for children with AIH, but not for those with ASC.

Vigorous Activation of Monocytes in Juvenile Autoimmune Liver Disease Escapes the Control of Regulatory T-Cells

Interface hepatitis, the histological lesion typical of autoimmune hepatitis (AIH), is composed of CD4 and CD8 T lymphocytes and of innate immunity cells, particularly monocytes. Studies in AIH have focused on autoreactive CD4 and CD8 T cells and impairment of CD4+CD25+ regulatory T cells (T‐regs), whereas little is known about the role of monocytes and their relationship with T‐regs. We have investigated 51 patients with autoimmune liver disease (AILD) and 27 healthy subjects, finding that monocytes were higher in number (P = 0.044), had a more vigorous spontaneous migration (P < 0.0005 in patients with inactive disease [ID], and P < 0.001 in those with active disease [AD]), displayed a higher tumor necrosis factor alpha (TNF‐α) over interleukin (IL)‐10 production (P = 0.07 in ID and P = 0.0005 in AD), and expressed higher levels of Toll‐like receptor (TLR) 4 (P = 0.048 in ID and P = 0.03 in AD). Addition of conventional T‐regs (cT‐regs) in AILD enhanced monocyte migration (P = 0.05 in ID and P = 0.08 in AD), magnified TNF‐α over IL‐10 production (P = 0.0005 in ID and P = 0.006 in AD), and markedly increased TLR4 expression levels (P = 0.01 in ID and P = 0.004 in AD), whereas in normal subjects it either restrained or left unchanged monocyte function. Because a CD127‐negative subpopulation within CD4+CD25+ T cells exerts the strongest regulatory activity, we performed additional experiments using purified CD4+CD25+CD127− T cells (true T‐regs [tT‐regs]). Addition of tT‐regs to monocytes decreased monocyte migration (P = 0.03) and promoted IL‐10 production (P = 0.009), leaving unchanged TLR4 expression in healthy subjects, whereas in patients with AILD it induced only a marginal increase in IL‐10 production (P = 0.045 in ID and P = 0.13 in AD). Conclusion: Monocyte overactivation and inability of cT‐regs and tT‐regs to restrain it may contribute to the loss of immune tolerance and perpetuation of the autoimmune attack in AILD. (HEPATOLOGY 2009.)

Cystatin C, an easy and reliable marker for assessment of renal dysfunction in children with liver disease and after liver transplantation

Renal dysfunction of variable severity is being increasingly recognized as a major complication of calcineurin inhibitors (CI), in some patients even necessitating renal transplantation. Close and effective monitoring of the renal function is indicated. Current methods for this monitoring are calculation of the glomerular filtration rate (GFR) based on creatinine or exogenous substances like 51Cr‐EDTA. The first method is unreliable in children and the second is expensive and cumbersome. Cystatin C has been shown to be an accurate marker of glomerular filtration but has not been evaluated in a large cohort of pediatric patients before and after liver transplantation (LT). We evaluated the accuracy of cystatin C in 62 children (30 male) with LT, who had their 51Cr‐EDTA measured on 40 occasions prior to LT and on 47 occasions after LT. The reciprocal of cystatin C correlated better with 51Cr‐EDTA GFR (r = .78) than the reciprocal of creatinine (r = .40). Diagnostic accuracy in the identification of reduced GFR was assessed by ROC analysis. Cystatin C yielded the highest area under the ROC curve (AUC) in all groups assessed. From these data a cutoff level of cystatin C predicting 51Cr‐EDTA GFR < 80 ml/min/1.73m2 was calculated. A level of 1.06 mg/L was found to have a sensitivity of 91% and a specificity of 81%. Applying this cutoff level in our patient group would have avoided 51Cr‐EDTA GFR estimation in 43 of the 87 estimations. In conclusion, the use of this simple test could be recommended as screening of renal dysfunction in children with liver disease and after LT. (Liver Transpl 2005;11:344–349.)

Liver abscesses in children: A single center experience in the developed world

Objectives: The aim of this study was to investigate the clinical and radiologic features, predisposing risk factors, and complications of children with pyogenic liver abscess (PLA) referred to a tertiary pediatric hepatology center. Methods: We analyzed our database of all children referred to our unit over a 10 year period and performed a case note review of all patients with a radiologically proven PLA. Results: PLA was diagnosed in 15 children (7 boys), 0.5% of all referrals. They presented at a median age of 10 years (range 2 months-15 years). In three children (2 boys), PLA was the first manifestation of chronic granulomatous disease. Among the others, five had radiologic evidence of other intra-abdominal pathology (1 with subsequently proven appendicitis), and four developed portal vein thrombosis with portal hypertension. The commonest isolated pathogen was Staphylococcus aureus. Combined treatment with guided aspiration and prolonged intravenous antibiotics was successful in all patients. Conclusion: PLA is a rare diagnosis in children in the developed world. It may be caused by primary neutrophil disorders even in the absence of a previous history of infection. Co-existent appendicitis, intra-abdominal sepsis, and ascending pylephlebitis must be sought because these children are at risk of developing portal vein obstruction and portal hypertension. Prolonged intravenous antibiotic treatment guided by microbiologic sensitivities is highly effective.

Auxiliary transplantation for acute liver failure: Histopathological study of native liver regeneration

Auxiliary liver transplantation (ALT) permits the serial assessment of regeneration in livers of patients with acute liver failure (ALF). Forty‐nine ALF patients [32 adults (median age, 23 years; range, 16‐40 years) and 17 children (median age, 12 years; range, 1‐15 years)] underwent ALT between 1994 and 2004 at Kings College Hospital. Twenty‐four patients had seronegative liver failure, 15 had acetaminophen toxicity, 4 had hepatitis B virus (HBV) infection, 3 had drug‐induced liver failure, 2 had autoimmune hepatitis, and 1 had mushroom poisoning. Nine patients without post‐ALT native liver histology were excluded from review. All acetaminophen‐induced, HBV, and drug‐related patients had diffuse injury. Twelve seronegative patients and the autoimmune hepatitis patient had a map‐like injury. On follow‐up, 9 acetaminophen‐induced patients, 9 seronegative patients, 2 drug‐induced ALF patients, 3 HBV patients, and the autoimmune patient recovered to a near‐normal native liver with inconsequential scarring. The hepatocyte proliferative rate in diffuse necrosis was 27.4% (range, 3.1%‐69.4%) at hepatectomy and sharply decreased after 8 days post‐ALT, being minimal months and years after ALT. In conclusion, in patients undergoing ALT for ALF with a diffuse pattern of liver injury—mainly acetaminophen toxicity—hepatocyte proliferation occurs in the native liver within a few days of transplantation. If the injury is map‐like (most cases of seronegative ALF), regeneration seems to involve variable hepatocellular proliferation and potential ductular hepatopoiesis, but sequential assessment is difficult because of sampling variation. The likelihood of histological recovery appears to be minimal in livers with total hepatocyte loss at the time of ALT. Liver Transpl 14:1437–1448, 2008.

Biliary atresia and other cholestatic childhood diseases: Advances and future challenges.

Biliary Atresia and other cholestatic childhood diseases are rare conditions affecting the function and/or anatomy along the canalicular-bile duct continuum, characterised by onset of persistent cholestatic jaundice during the neonatal period. Biliary atresia (BA) is the most common among these, but still has an incidence of only 1 in 10-19,000 in Europe and North America. Other diseases such as the genetic conditions, Alagille syndrome (ALGS) and Progressive Familial Intrahepatic Cholestasis (PFIC), are less common. Choledochal malformations are amenable to surgical correction and require a high index of suspicion. The low incidence of such diseases hinder patient-based studies that include large cohorts, while the limited numbers of animal models of disease that recapitulate the spectrum of disease phenotypes hinders both basic research and the development of new treatments. Despite their individual rarity, collectively BA and other cholestatic childhood diseases are the commonest indications for liver transplantation during childhood. Here, we review the recent advances in basic research and clinical progress in these diseases, as well as the research needs. For the various diseases, we formulate current key questions and controversies and identify top priorities to guide future research.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Implementation of the Lancet Standing Commission on Liver Disease in the UK

Between 1980 and 2013, deaths from liver disease in the UK increased by four times, mainly attributable to alcohol consumption. Although the latest data from Public Health England (PHE) show some reduction in the number of hospital admissions for alcohol-related liver disease in those younger than 18 years and in the number drinking, the high mortality in adults continues with 50% of English local authorities seeing a rise in hospital admissions for alcohol-related illness. Obesity-related liver disease and prevalence of primary hepatocellular carcinoma (HCC) are both increasing, with ever increasing costs to the National Health Service (NHS). The ten major recommendations in the Lancet Commission report were selected as needing urgent implementation on the basis of strong evidence and are considered in terms of what has been achieved to date and where there is ongoing work.

Should we retransplant a patient who is non‐adherent? A literature review and critical reflection

Dobbels F, Hames A, Aujoulat I, Heaton N, Samyn M. Should we retransplant a patient who is non‐adherent? A literature review and critical reflection. 
Pediatr Transplantation 2012: 16: 4–11.