Giorgina Mieli-Vergani

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and γ-glutamyltranspeptidase (γGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

Diagnosis and management of autoimmune hepatitis

Clinical practice guidelines are defined as ‘‘systematically developed statements to assist practitioner and patient decisions about appropriate heath care for specific clinical circumstances.’’ (All references are available in the Supporting Information.) These guidelines on autoimmune hepatitis provide a data-supported approach to the diagnosis and management of this disease. They are based on the following: (1) formal review and analysis of the recently-published world literature on the topic [Medline search]; (2) American College of Physicians Manual for Assessing Health Practices and Designing Practice Guidelines; (3) guideline policies, including the AASLD Policy on the Development and Use of Practice Guidelines and the American Gastroenterological Association Policy Statement on Guidelines; and (4) the experience of the authors in the specified topic. These recommendations, intended for use by physicians, suggest preferred approaches to the diagnostic, therapeutic and preventive aspects of care. They are intended to be flexible, in contrast to standards of care, which are inflexible policies to be followed in every case. Specific recommendations are based on relevant published information. To more fully characterize the quality of evidence supporting the recommendations, the Practice Guidelines Committee of the AASLD requires a class (reflecting benefit versus risk) and level (assessing strength or certainty) of evidence to be assigned and reported with each recommendation. The grading system applied to the recommendations has been adapted from the American College of Cardiology and the American Heart Association Practice Guidelines, and it is given below (Table 1).

De-novo autoimmune hepatitis after liver transplantation.

Summary Background Late graft dysfunction that does not result from recognised causes, such as rejection, infection, or vascular or biliary complications, can occur after liver transplantation. We investigated a particular type of unexplained graft dysfunction that is associated with autoimmune features in children who underwent transplantation at our unit between 1991 and 1996. Methods Seven (4%) of 180 liver-transplant recipients developed an unexplained but characteristic form of graft dysfunction (five boys, two girls; median age at presentation 10·3 years, range 2·0–19·4). The median period after surgery was 24 months (6–45). The indications for transplantation had been extrahepatic biliary atresia (four patients), Alagilles syndrome (one), drug-induced acute liver failure (one), and α1-antitrypsin deficiency (one). Four patients were on triple immunosuppression with cyclosporin, azathioprine, and prednisolone; and three were on tacrolimus. Immunoglobulin measurements, autoantibody studies, serological studies, and HLA typing were undertaken. Liver-biopsy samples were taken. Findings Infectious and surgical complications were excluded. Liver-biopsy samples showed the histological changes of chronic hepatitis, including portal and periportal hepatitis with lymphocytes and plasma cells, bridging collapse, and perivenular-cell necrosis without changes typical of acute or chronic rejection. All patients had high concentrations of IgG (median 22 g/L [range 17·2–34·4]) and high titres of autoantibodies. All but one patient responded to prednisolone 2 mg/kg daily and an increase in or addition of azathioprine (1·5 mg/kg daily) within a median of 32 days (7–316). One responder relapsed owing to poor compliance but went into remission after treatment was restored. All six respondents remain in remission on a reduced dose of prednisolone (5–10 mg/day) and 1·5 mg/kg daily azathioprine at a median of 283 days (range 108–730) follow-up. Interpretation Our data show that symptoms of autoimmune hepatitis, which are responsive to the classical treatment for this condition, can appear in liver-transplant patients while they are on anti-rejection immunosuppression. Whether the liver damage in these patients is a form of rejection or the consequence of autoimmune attack has yet to be established.

Functional Study of CD4+CD25+ Regulatory T Cells in Health and Autoimmune Hepatitis

Regulatory CD4+CD25+ T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-γ production by CD4+CD25− and CD8+ T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-β, suggesting a mechanism of linked immunosuppression. Tregs/CD4+CD25− T cell cocultures lead to similar changes in IFN-γ and IL-10 secretion in patients and controls, whereas increased TGF-β secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8+ T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4+CD25− T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4+CD25− T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4+CD25− T cell spontaneous apoptosis may contribute to the development of autoimmunity.

Antibodies to the Surface of Halothane-Altered Rabbit Hepatocytes in Patients with Severe Halothane-Associated Hepatitis

Circulating antibodies reacting specifically with the cell membrane of hepatocytes isolated from halothane-anesthetized rabbits were detected in nine of 11 patients with fulminant hepatic failure after helothane-induced anesthesia. The immunoglobulin deposition, as revealed by immunofluorescence, showed a granular pattern on the hepatocyte surface membrane. Preincubation of halothane-pretreated, but not of control, hepatocytes with serum containing this antibody rendered them susceptible to cytotoxic effects of normal lymphocytes in vitro. Control studies using serum from subjects repeatedly exposed to halothane without the development of liver damage, and from patients with viral and toxic liver injury have confirmed the specificity of these findings to serve halothane-associated liver injury. These results provide further evidence of an immunologic component in this condition.

Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency

Hepatocellular carcinoma (HCC) is rare in young children. We attempted to see if immunohistochemical and mutational‐analysis studies could demonstrate that deficiency of the canalicular bile acid transporter bile salt export pump (BSEP) and mutation in ABCB11, encoding BSEP, underlay progressive familial intrahepatic cholestasis (PFIC)—or “neonatal hepatitis” suggesting PFIC—that was associated with HCC in young children. We studied 11 cases of pediatric HCC in the setting of PFIC or “neonatal hepatitis” suggesting PFIC. Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11 was performed in leukocyte DNA from available patients and parents. Among the 11 nonrelated children studied aged 13‐52 months at diagnosis of HCC, 9 (and a full sibling, with neonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) had immunohistochemical evidence of BSEP deficiency; the eleventh child did not. Mutations in ABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNA could be studied (n = 7). These mutations were confirmed in the parents (n = 14). With respect to the other 3 children with BSEP deficiency, mutations in ABCB11 were demonstrated in all 5 parents in whom leukocyte DNA could be studied. Thirteen different mutations were found. In conclusion, PFIC associated with BSEP deficiency represents a previously unrecognized risk for HCC in young children. Immunohistochemical evidence of BSEP deficiency correlates well with demonstrable mutation in ABCB11. (HEPATOLOGY 2006;44:478–486.)

Severe bile salt export pump deficiency : 82 different ABCB11 mutations in 109 families

BACKGROUND & AIMS Patients with severe bile salt export pump (BSEP) deficiency present as infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy. METHODS Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome. RESULTS Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7 families, only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those carrying 2 null mutations, is essential.

Wilson's disease in children: 37‐Year experience and revised King's score for liver transplantation

Wilsons disease (WD) is a rare liver‐based disorder of copper metabolism. Prognostic criteria described by our group in 1986 to predict death without transplantation have not been universally validated. The clinical features of 88 children were reviewed, retrospectively in 74 and prospectively in 14. Data from the retrospectively recruited patients that died or survived on long‐term chelation were used to evaluate the validity of our old scoring system and to devise a new prognostic index, then assessed in the 14 prospectively recruited patients. Using the old scoring system, 5 children scoring ≥ 7, the cutoff value for death without transplantation, survived, whereas 4 scoring ≤ 7 died (sensitivity 87% and specificity 90%). A new index based on serum bilirubin, international normalized ratio, aspartate aminotransferase (AST), and white cell count (WCC) at presentation identified a cutoff score of 11 for death and proved to be 93% sensitive and 98% specific, with a positive predictive value of 88%. When the new index was evaluated prospectively in 14 patients, it predicted the need for transplantation in only the 4 who required it, although 1 child with a score of 11 survived on medical treatment. In conclusion, the new Wilson Index is more sensitive and specific in predicting mortality without transplantation than the old scoring system, but needs to be validated in a larger number of patients. (Liver Transpl 2005;11:441–448.)

Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered questions

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

LATE REFERRAL FOR BILIARY ATRESIA— MISSED OPPORTUNITIES FOR EFFECTIVE SURGERY

To assess whether clinicopathological features other than the age at operation influence prognosis after surgery for extrahepatic biliary atresia (EHBA) and to determine whether the age at referral has fallen since a previous survey, 50 consecutive cases with EHBA referred between February, 1985, and December, 1987, were reviewed. Liver or spleen size, liver function tests, or histological appearance of liver biopsy specimen before surgery were not predictive of outcome. The jaundice cleared up in 12 of 14 children operated on by age 8 weeks, but in only 13 of 36 operated on later. In 41 referral was delayed. All 25 children in whom surgery was successful are alive and well, while 13 of 25 with unsuccessful surgery have died, at a median age of 1 year. To improve the prognosis of infants with EHBA parents and health staff need a better awareness of the early clinical features of EHBA and of the necessity for prompt referral. Liver disease should be suspected in any infant jaundiced after 14 days of age.