Heinz Zoller

MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.

Following homozygosity mapping in a single kindred, we identified nonsense and missense mutations in MYO5B, encoding type Vb myosin motor protein, in individuals with microvillus inclusion disease (MVID). MVID is characterized by lack of microvilli on the surface of enterocytes and occurrence of intracellular vacuolar structures containing microvilli. In addition, mislocalization of transferrin receptor in MVID enterocytes suggests that MYO5B deficiency causes defective trafficking of apical and basolateral proteins in MVID.

Anti-tumor necrosis factor-alpha monoclonal antibody therapy in severe alcoholic hepatitis

BACKGROUND/AIMS Severe alcoholic hepatitis (AH) is associated with high mortality. Tumor necrosis factor-alpha (TNFalpha) has been demonstrated to play an important role in its pathophysiology. METHODS Twelve patients with biopsy-confirmed AH and a Maddrey discriminant factor >32 were treated with a single infusion of the anti-TNF monoclonal antibody Infliximab at a dose of 5mg/kg body weight. Serial measurements were made for various cytokines using specific enzyme-linked immunoassays (ELISA). In four patients, liver biopsy samples were available pretreatment and on day+28 of therapy. RESULTS Ten of the 12 patients are alive at a median of 15 (12-20) months. Two patients died within 30 days from septicemia. Serum bilirubin levels, Maddrey score, neutrophil count and C-reactive protein fell significantly within the first month. There was an early, though not significant, decrease in plasma levels of proinflammatory cytokines (interleukins (IL)-1beta, IL-6, IL-8, interferon-gamma), whereas plasma levels of TNFalpha remained near the sensitivity limit of the assay throughout the treatment course. While TNFalpha mRNA expression in the liver did not change, expression of IL-8, a cytokine regulated mainly by TNFalpha, was almost absent on day+28. CONCLUSIONS Our data suggest that randomized controlled trials of anti-TNF antibody in severe AH are warranted.

Duodenal metal-transporter (DMT-1, NRAMP-2) expression in patients with hereditary haemochromatosis.

BACKGROUND Although the gene for hereditary haemochromatosis has been cloned, the mechanism by which iron uptake is inappropriately increased in this disorder is unclear. Iron absorption is regulated by the duodenal metal transporter, DMT-1, also called NRAMP-2. We investigated the expression of NRAMP-2 in patients with hereditary haemochromatosis. METHODS Duodenal biopsy samples were taken from 20 patients with haemochromatosis homozygous for the C282Y mutation and from ten controls. NRAMP-2 expression was assessed by northern blotting and competitive PCR. NRAMP-2 mRNA was sequenced in seven patients and two controls. FINDINGS Duodenal NRAMP-2 mRNA concentrations were increased in patients as estimated by Northern blotting. Accordingly, competitive PCR showed significantly higher NRAMP-2 cDNA concentrations in patients than in controls (mean 3.43 [SD 0.61] vs 1.11 [0.74] log ng competitor x 10(4); p<0.001). No mutations were found within the NRAMP-2 mRNA. Duodenal NRAMP-2 mRNA expression was correlated with serum ferritin in controls (r=-0.94, p=0.001) but not in patients (r=-0.18, p=0.8). INTERPRETATION Increased NRAMP-2 mRNA expression in duodenal mucosa of patients with hereditary haemochromatosis may promote duodenal iron uptake and lead to iron overload.

Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells

In order to examine the regulatory effects of major Th1‐derived cytokines, such as IL‐12, and Th2 cytokines, IL‐4 and IL‐10, on the formation of neopterin and degradation of tryptophan, two metabolic pathways induced by interferon‐gamma (IFN‐γ) in human monocytes/macrophages, we investigated the human monocytic cell line THP‐1, primary human macrophages, and peripheral blood mononuclear cells (PBMC). Neopterin formation and tryptophan degradation were induced similarly by IFN‐γ in all three cell types investigated, but the effects of interleukins were different between THP‐1, primary macrophages and PBMC. In PBMC, but not in THP‐1 cells and primary macrophages, IL‐12 was found to be additive to the effects of IFN‐γ to superinduce neopterin formation and tryptophan degradation. IL‐4 and IL‐10 reduced the effects of IFN‐γ on monocytic cells, and both cytokines were additively antagonistic to IFN‐γ in PBMC and THP‐1 cells. Finally, on preincubation, but not on addition of IL‐12, the effects of IL‐4 and IL‐10 on PBMC could be abrogated, whereas no such effect was seen in THP‐1 cells. The results show that IL‐12 up‐regulates neopterin formation and tryptophan degradation by inducing additional IFN‐γ production by Th1 cells, while a direct effect of IL‐12 on monocytes/macrophages appears to be absent. Similarly, IL‐4 and IL‐10 inhibit neopterin production and tryptophan degradation in PBMC by down‐regulating Th1‐type cytokine production and possibly also via direct deactivation of IFN‐γ effects towards monocytes/macrophages. The results clearly show how Th1 cell‐mediated immunity may be up‐ or down‐regulated by endogenous cytokine production.

Ferroportin disease: A systematic meta-analysis of clinical and molecular findings

Background & Aims Classical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies. Methods We conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease. Results Of the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms. Conclusions In contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron.

Acute-on-chronic liver failure: excellent outcomes after liver transplantation but high mortality on the wait list.

Acute‐on‐chronic liver failure (ACLF) is characterized by high short‐term mortality. Liver transplantation (LT) is a potential therapy for patients who do not improve with supportive measures, but the efficacy of LT has not been shown. The aim of this study was to investigate the feasibility of LT and to determine the postoperative outcomes of patients with ACLF. All patients referred to our liver unit between 2002 and 2010 were registered in a database. The diagnosis of ACLF was made in accordance with the Asian Pacific Association for the Study of the Liver consensus. The post‐LT outcomes were compared with the outcomes of a cohort of patients with chronic liver disease who underwent transplantation for other indications during the same period. One hundred forty four of 238 patients fulfilled the ACLF criteria. In an intention‐to‐treat analysis, the median transplant‐free survival time was 48 days. Multiorgan failure was the most common cause of death. Ninety‐four patients (65%) were evaluated for LT, 71 patients (49%) were listed, and 33 patients (23%) finally underwent deceased donor LT; this resulted in a wait‐list mortality rate of 54%. Patients who developed infectious complications (particularly pneumonia and/or sepsis) and patients who received renal replacement therapy or mechanical ventilation were less likely to undergo LT. The 1‐ and 5‐year survival rates of 87% and 82% were comparable to the rates for non‐ACLF patients. In conclusion, this study shows that LT remains the only therapeutic option for the vast majority of patients with ACLF. However, LT was feasible in less than one fourth of the patients with a 5‐year survival rate greater than 80%. Liver Transpl 19:879‐886, 2013.

Efficacy and Safety of Oral Chelators in Treatment of Patients With Wilson Disease

BACKGROUND & AIMS Wilson disease is a genetic copper storage disorder that causes hepatic and neurologic symptoms. Chelating agents (D-penicillamine, trientine) are used as first-line therapies for symptomatic patients, but there are few data from large cohorts. We assessed the safety of D-penicillamine and trientine therapy and outcomes of patients with Wilson disease. METHODS We performed a retrospective analysis of data on 380 patients with Wilson disease from tertiary care centers in Germany and Austria, and 25 additional patients from the EUROWILSON registry. Chelator-based treatment regimens were analyzed for their effect on neurologic and hepatic symptoms and for adverse events that led to discontinuation of therapy (Kaplan-Meier estimation; data were collected for a mean of 13.3 y after therapy began). RESULTS Changes in medication were common, resulting in analysis of 471 chelator monotherapies (326 patients receiving D-penicillamine and 141 receiving trientine). Nine of 326 patients treated with D-penicillamine and 3 of 141 patients given trientine underwent liver transplantation. Adverse events leading to discontinuation of treatment were more frequent among those receiving D-penicillamine than trientine (P = .039). Forty-eight months after therapy, hepatic deterioration was reported in only 4 of 333 patients treated initially with a chelating agent. Hepatic improvements were observed in more than 90%, and neurologic improvements were observed in more than 55%, of therapy-naive patients, and values did not differ significantly between treatments. However, neurologic deterioration was observed less frequently in patients given D-penicillamine first (6 of 295) than those given trientine first (4 of 38; P = .018). CONCLUSIONS Chelating agents are effective therapies for most patients with Wilson disease; D-penicillamine and trientine produce comparable outcomes, although D-penicillamine had a higher rate of adverse events. Few patients receiving chelation therapy had neurologic deterioration, which occurred more frequently in patients who received trientine.

Mutations in SPINT2 Cause a Syndromic Form of Congenital Sodium Diarrhea

Autosomal-recessive congenital sodium diarrhea (CSD) is characterized by perinatal onset of a persistent watery diarrhea with nonproportionally high fecal sodium excretion. Defective jejunal brush-border Na(+)/H(+) exchange has been reported in three sporadic patients, but the molecular basis of the disease has not been elucidated. We reviewed data from a large cohort of CSD patients (n = 24) and distinguished CSD associated with choanal or anal atresia, hypertelorism, and corneal erosions--i.e., a syndromic form of CSD--occurring in ten families from an isolated form--i.e., classic CSD--presenting in seven families. Patients from both groups have a high risk of mortality due to immediate electrolyte imbalances and complications from long-term parenteral nutrition in the first years of life, but survivors can eventually adapt to partial or complete enteral nutrition. A genome-wide SNP scan was applied and identified a homozygous c.593-1G-->A splicing mutation in SPINT2, encoding a Kunitz-type serine-protease inhibitor, in one extended kindred with syndromic CSD. The same mutation and four distinct, homozygous or compound heterozygous mutations (p.Y163C, c.1A-->T, c.337+2T-->C, c.553+2T-->A) were identified in all syndromic patients. No SPINT2 mutations were found in classic-CSD patients. SPINT2 mutations were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro. We delineate syndromic CSD as a distinct disease entity caused by SPINT2 loss-of-function mutations. SPINT2 mutations might lead to an excess of yet unknown serine protease activity in affected tissues.

Duodenal cytochrome B and hephaestin expression in patients with iron deficiency and hemochromatosis

BACKGROUND & AIMS An increased duodenal expression of the iron transporters, divalent-metal-transporter-1, and ferroportin is observed in patients with iron deficiency or hereditary hemochromatosis. Two oxidoreductases, termed duodenal cytochrome b and hephaestin, are proposed to co-operate with divalent-metal-transporter-1 and FPN1, respectively, to transfer iron from the duodenal lumen to the circulation. METHODS In the present study, we investigated the mRNA and protein expression of Dcytb and hephaestin in duodenal biopsies from patients with iron deficiency, HFE, and non-HFE-associated hemochromatosis and in control subjects by means of real-time polymerase chain reaction, Western blot, and immunofluorescence. RESULTS In iron deficiency a coordinated upregulation of the iron transporters divalent-metal-transporter-1 and ferroportin and of duodenal-cytochrome b and hephaestin was found, whereas in patients with HFE and non-HFE-associated hemochromatosis duodenal-cytochrome b and hephaestin protein and mRNA expression were not significantly different from control subjects. However, HFE but not non-HFE hemochromatosis patients presented with an increased duodenal ferric reductase activity. Spearman rank correlations showed that Dcytb, hephaestin, FPN1, and DMT1 mRNA expression are positively related to each other independently of the underlying disease, which ensures an efficient transepithelial transport of absorbed iron. CONCLUSIONS Our data show that duodenal-cytochrome b activity in iron deficiency is stimulated via enhanced protein expression, whereas in HFE hemochromatosis it is up-regulated post-translationally. This points to different kinetics of intestinal iron uptake between iron deficiency and HFE hemochromatosis and also indicates that duodenal iron accumulation in HFE and non-HFE hemochromatosis is pathophysiologically different.

Iron regulates hepatitis C virus translation via stimulation of expression of translation initiation factor 3

BACKGROUND Although the response to treatment with interferon- alpha in individuals with chronic hepatitis C virus (HCV) infection is negatively associated with increased liver iron stores, the underlying mechanisms at work have remained elusive to date. The translation initiation factor 3 (eIF3) is essential for HCV translation, and thus the effects that iron perturbations have on eIF3 expression and HCV translation were studied here. METHODS eIF3 expression was analyzed by TaqMan polymerase chain reaction, Northern and Western blot analysis of HepG2 cells, and liver biopsies. Functional effects of iron on HCV mRNA translation were estimated by use of transient transfection experiments with bicistronic vectors. RESULTS Iron treatment of HepG2 cells increased eIF3 mRNA and protein expression, whereas iron chelation reduced it. Accordingly, iron-dependent stimulation of eIF3 specifically induced the expression of reporter genes under the control of regulatory HCV mRNA stem-loop structures. Moreover, a positive association between liver iron levels, eIF3 expression, and HCV expression was found when liver-biopsy samples from HCV-infected patients were analyzed. CONCLUSION Iron promotes the translation of HCV by stimulating the expression of eIF3, which may be one reason for the negative association between liver iron overload and HCV infection. Modulation of the affinity of eIF3 to bind to HCV mRNA may be a promising target for the treatment of chronic HCV infection.