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PurposeCOViK - a prospective hospital-based multicenter case-control study in Germany - aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave.MethodsWe analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates.Results21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses.ConclusionThree vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.Ethical review and trial registrationThe study was approved by the Ethics Committee of the Charité Universitätsmedizin, Berlin (EA1/063/21) and was registered at “Deutsches Register Klinischer Studien” (DRKS00025004).Potential conflicts of interestS. G. received payment/honoraria from Astra Zeneca, Boehringer Ingelheim, Roche Pharma, Novartis and Berlin Chemie, this had no influence on this work; all other authors reported no conflicts of interest.

Wilson disease (WD) is a multisystem disorder that manifests primarily with hepatic and neurological symptoms due to copper toxicity in liver and brain. More than two decades after identification of the defective gene, ATP7B, the underlying mechanisms of copper toxicity and the striking phenotypic diversity remain far from understood. Animal models with defects in the Atp7b homolog have provided new insights into pathophysiology of WD. Several rodent models harboring Atp7b defects have been described, which include inbred mouse models [toxic milk [tx; tx-r] mouse, Jackson tx [tx-j] mouse], the Atp7b−/− (knockout) mouse, as well as the Long-Evans Cinnamon and LPP rats, all of which develop liver disease. Brain copper accumulation accompanied by neurologic involvement was observed in several of these animals; however, symptoms were less severe compared to humans. These models are valuable for exploration of copper metabolism, gene expression, and investigation of WD progression. To facilitate mandatory new research on novel drugs, gene, and cell therapy, animal models constitute an invaluable tool.

UNLABELLED Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. CONCLUSIONS The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841).

Background & Aims Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver—the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues. Methods We collected intestinal tissues from ATP7B-knockout (Atp7b −/− ) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy. Results ATP7B maintains a Cu gradient along the duodenal crypt−villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b −/− mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons. Conclusions ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.

Wilson disease (WD) is caused by ATPase copper-transporting beta (ATP7B) mutations and results in copper toxicity in liver and brain. Although the defective gene was identified in 1993, the specific mechanisms underlying copper toxicity and the remarkable phenotypic diversity of the disease are still poorly understood. Animal models harboring defects in the ATP7B homolog have helped to reveal new insights into pathomechanisms of WD. Four rodent models with ATP7B gene defects have been described - the Long-Evans Cinnamon (LEC) rat, inbred mouse models (toxic milk (tx), the Jackson Laboratory toxic milk (tx-j)), and the genetically engineered ATP7B

Wilson's disease (WD) is caused by ATP7B mutations and results in copper accumulation and toxicity in liver and brain tissues. The specific mechanisms underlying copper toxicity are still poorly understood. Mouse models have revealed new insights into pathomechanisms of hepatic WD. Mitochondrial damage is observed in livers of WD patients and in mouse models; copper induces fragmentation of mitochondrial membrane lipids, particularly cardiolipin, with deleterious effects on both mitochondrial integrity and function. Copper accumulation also induces chronic inflammation in WD livers, which is followed by regeneration in parts of the liver and occasionally neoplastic proliferation. Gene expression studies using microarrays have aided our understanding of the molecular basis of these changes. Copper overload alters cholesterol biosynthesis in hepatocytes resulting in reduced liver and serum cholesterol. Experiments are currently underway to elucidate the link between copper and cholesterol metabolism. These findings may facilitate the development of specific therapies to ameliorate WD progression.

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b(-/-) mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu(2+)/H(2)O(2)/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b(-/-) mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b(-/-) mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b(-/-) mice in contrast to control with increasing age. Hepatocytes from elder Atp7b(-/-)mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b−/− hepatocytes, but reaches a limit at 90–300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.

Background— Phytosterols are plant-derived sterols that are taken up from food and can serve as biomarkers of cholesterol uptake. Serum levels are under tight genetic control. We used a genomic approach to study the molecular regulation of serum phytosterol levels and potential links to coronary artery disease (CAD). Methods and Results— A genome-wide association study for serum phytosterols (campesterol, sitosterol, brassicasterol) was conducted in a population-based sample from KORA (Cooperative Research in the Region of Augsburg) (n=1495) with subsequent replication in 2 additional samples (n=1157 and n=1760). Replicated single-nucleotide polymorphisms (SNPs) were tested for association with premature CAD in a metaanalysis of 11 different samples comprising 13 764 CAD cases and 13 630 healthy controls. Genetic variants in the ATP-binding hemitransporter ABCG8 and at the blood group ABO locus were significantly associated with serum phytosterols. Effects in ABCG8 were independently related to SNPs rs4245791 and rs41360247 (combined P =1.6×10 −50 and 6.2×10 −25 , respectively; n=4412). Serum campesterol was elevated 12% for each rs4245791 T-allele. The same allele was associated with 40% decreased hepatic ABCG8 mRNA expression ( P =0.009). Effects at the ABO locus were related to SNP rs657152 (combined P =9.4×10 −13 ). Alleles of ABCG8 and ABO associated with elevated phytosterol levels displayed significant associations with increased CAD risk (rs4245791 odds ratio, 1.10; 95% CI, 1.06 to 1.14; P =2.2×10 −6 ; rs657152 odds ratio, 1.13; 95% CI, 1.07 to 1.19; P =9.4×10 −6 ), whereas alleles at ABCG8 associated with reduced phytosterol levels were associated with reduced CAD risk (rs41360247 odds ratio, 0.84; 95% CI, 0.78 to 0.91; P =1.3×10 −5 ). Conclusion— Common variants in ABCG8 and ABO are strongly associated with serum phytosterol levels and show concordant and previously unknown associations with CAD.

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H(2)O(2)/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H(2)O(2)/ascorbate or with Cu(2+)/H(2)O(2)/ascorbate.

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene, ATP7B, has been identified, but the molecular events leading to Wilson disease remain poorly understood. Here, we demonstrate that genetically engineered Atp7b−/− mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of Atp7b−/− livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with Atp7b inactivation.

Der Morbus Wilson ist eine autosomal-rezessiv vererbte Kupferstoffwechselstorung, die mit neurologischen Symptomen und Leberschadigung unterschiedlichen Ausmases einhergeht. Das betroffene Gen ATP7B kodiert fur ein hepatisches Kupfertransportprotein mit zentraler Rolle im Kupferstoffwechsel des Menschen. Das klinische Bild ist komplex, neben einer vorwiegend neurologischen Verlaufsform mit Tremor, Sprachstorungen, psychischen Veranderungen etc., werden vorwiegend hepatische Verlaufe und Mischformen beobachtet. In der Leber kann die Kupfereinlagerung zu akutem Leberversagen, chronischer Hepatitis oder Leberzirrhose fuhren. Fruhes Erkennen der Erkrankung durch klinische, biochemische und genetische Untersuchung und die rasche Therapieeinleitung mit Chelatoren oder Zinksalzen sind fur Verlauf und Prognose des Morbus Wilson entscheidend. Die Lebertransplantation stellt eine Alternative bei akutem oder chronischen Leberversagen dar und fuhrt zur Heilung der Lebererkrankung. Verlaufskontrollen zum Erkennen von relativ haufigen Therapienebenwirkungen, Uberwachung der Patienten-Compliance und des Therapieerfolgs sollten individuell und engmaschig erfolgen.

Background & Aims: In nonrandomized trials, photodynamic therapy (PDT) had a promising effect on nonresectable cholangiocarcinoma (NCC). This prospective, open-label, randomized, multicenter study with a group sequential design compared PDT in addition to stenting (group A) with stenting alone (group B) in patients with NCC. Methods: In patients with histologically confirmed cholangiocarcinoma, endoscopic or percutaneous double stenting was performed. Patients fulfilling inclusion criteria were randomized to group A (stenting and subsequent PDT) and group B (stenting alone). For PDT, Photofrin 2 mg/kg body wt was injected intravenously 2 days before intraluminal photoactivation (wavelength, 630 nm; light dose, 180 J/cm 2 ). Further treatments were performed in cases of residual tumor in the bile duct. The primary outcome parameter was survival time. Secondary outcome parameters were cholestasis and quality of life. Results: PDT resulted in prolongation of survival (group A: n=20, median 493 days; group B: n=19, median 98 days; P Conclusions: PDT, given in addition to best supportive care, improves survival in patients with NCC. The study was terminated prematurely because PDT proved to be so superior to simple stenting treatment that further randomization was deemed unethical.

Wilson's disease (WD) is caused by ATP7B mutations and results in copper accumulation and toxicity in liver and brain tissues. The specific mechanisms underlying copper toxicity are still poorly understood. Mouse models have revealed new insights into pathomechanisms of hepatic WD. Mitochondrial damage is observed in livers of WD patients and in mouse models; copper induces fragmentation of mitochondrial membrane lipids, particularly cardiolipin, with deleterious effects on both mitochondrial integrity and function. Copper accumulation also induces chronic inflammation in WD livers, which is followed by regeneration in parts of the liver and occasionally neoplastic proliferation. Gene expression studies using microarrays have aided our understanding of the molecular basis of these changes. Copper overload alters cholesterol biosynthesis in hepatocytes resulting in reduced liver and serum cholesterol. Experiments are currently underway to elucidate the link between copper and cholesterol metabolism. These findings may facilitate the development of specific therapies to ameliorate WD progression.

Selective antibodies were used to assess the cellular and subcellular localization of glutathione, and the glutathione precursors γ-glutamylcysteine, glutamate, and cysteine, in neuronal (photoreceptors) and non-neuronal (pigment epithelial cells and Muller cells) cell types in the outer retina of the guinea pig. In each cell type the highest level of glutathione immunoreactivity occurred in the mitochondria. The labeling density in the cytoplasmic matrix was higher (and the mitochondrial-cytoplasmic gold particle ratio lower) in pigment epithelial cells than in Muller cells and photoreceptors. The latter two cell types showed a mitochondrial-cytoplasmic gold particle ratio of 15.5 and 21.7, respectively. In contrast to glutathione, γ-glutamylcysteine seemed to be enriched in the cytoplasmic matrix relative to the mitochondria. The immunogold labeling for this dipeptide was stronger in the pigment epithelial cells than in Muller cells and photoreceptors. Glutamate immunoreactivity was high in photoreceptors, intermediate in pigment epithelial cells, and low in Muller cells, while the cysteine immunogold signal was low in each cell type and cell compartment. The present results suggest that glutathione is concentrated in mitochondria but to different degrees in different cells. The low mitochondrial content of γ-glutamylcysteine (the direct precursor of glutathione) is consistent with biochemical data indicating that glutathione is synthesized extramitochondrially and transported into the mitochondrial matrix. Judged from the immunocytochemical data, cysteine may be a rate-limiting factor in glutathione synthesis in each cell type while glutamate can be rate limiting only in Muller cells.

Background & Aims Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. Methods We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. Results Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. Conclusions Variants in ATP7B associated with Wilson disease disrupt the protein's transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b(-/-) hepatocytes, but reaches a limit at 90-300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.

Wilson disease is an inherited autosomal recessive disorder of copper balance leading to hepatic damage and neurological disturbance of variable degree. The defective gene, ATP7B, encodes a hepatic copper-transporting protein, which plays a key role in human copper metabolism. Our knowledge of the genetic basis of Wilson disease has increased dramatically; however, understanding of genotype-phenotype correlation and multifarious effects of copper toxicity as basis for targeted and individualised therapy strategies is still insufficient. Clinical manifestations are related to copper accumulation predominantly in the liver and brain and include hepatic disease ranging from mild hepatitis to acute liver failure or cirrhosis and/or neurological symptoms such as dystonia, tremor, dysarthria, psychiatric disturbances. Mixed presentations occur frequently. Early recognition by means of clinical, biochemical or genetic examination and initiation of therapy with copper chelators, zinc salts or even liver transplantation in cases of acute and chronic liver failure are essential for favourable outcome.

Wilson disease (WD) is a disorder of copper transport caused by mutations within the ATP7B gene. WD is phenotypically variable and can present with predominantly hepatic or neurologic manifestations. The mechanisms responsible for this variability are unknown. GP73, a Golgi membrane protein, is expressed in hepatocytes in response to acute and chronic liver disease.Hepatocyte GP73 expression was examined in the livers of WD patients by semiquantitative immunohistochemistry. GP73 mRNA levels were measured in mice with a deletion of the WD gene (Atp7b(-/-)) by real-time PCR, and these values were compared to the concomitant histological abnormalities and previously reported copper levels.Hepatocyte GP73 expression was more frequently observed in patients with hepatic versus neurologic presentation (79% vs. 30%, p0.05). Furthermore, GP73 expression was significantly higher (44.7+/-14.0 vs. 2.0+/-0.81, p0.05) in patients with hepatic phenotype. In Atp7b(-/-) mice, GP73 mRNA was significantly elevated at 20-46 weeks of age, coincident with extensive hepatic inflammation and fibrosis, but not at 6 weeks, when hepatic histology was normal despite significant copper overload. GP73 mRNA levels normalized concomitantly with the resolution of hepatic injury at 60-weeks. However, in tumor-like nodules GP73 was strikingly elevated.Increased hepatocyte GP73 expression is more commonly a feature of hepatic than neurologic WD, and is triggered in response to inflammation, fibrosis, and dysplasia, rather than copper overload.

Cisplatin is a widely used chemotherapeutic agent for treatment of ovarian, testicular, lung, and stomach cancers. The initial response to the drug is robust; however, tumor cells commonly develop resistance to cisplatin, which complicates treatment. Recently, overexpression of the Cu-ATPase ATP7B in ovary cells was linked to the increased cellular resistance to cisplatin; and the role for Cu-ATPases in the export of cisplatin from cells was proposed. Our results support functional interactions between cisplatin and ATP7B but argue against the active transport through the copper translocation pathway as a mechanism of drug resistance. In hepatocytes, we observed no correlation between the levels of endogenous ATP7B and the resistance of cells to cisplatin. Unlike copper, cisplatin does not induce trafficking of ATP7B in hepatoma cells, neither does it compete with copper in a transport assay. However, cisplatin binds to ATP7B and stimulates catalytic phosphorylation with EC50 similar to that of copper. Mutations of the first five N-terminal copper-binding sites of ATP7B do not inhibit the cisplatin-induced phosphorylation of ATP7B. In contrast, the deletion of the first four copper-binding sites abolishes the effect of cisplatin on the ATP7B activity. Thus, cisplatin binding to ATP7B and/or general changes in cellular copper homeostasis are likely contributors to the increased resistance to the drug. The link between changes in copper homeostasis and cisplatin resistance was confirmed by treating the Huh7 cells with copper chelator and increasing their resistance to cisplatin.cisplatin

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu 2+ (Fe 2+ )/H 2 O 2 /ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu 2+ /H 2 O 2 /and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO -radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.

This is a case report of a 36 years old man who has been suffering for 20 years from benign recurrent intrahepatic cholestasis (BRIC). BRIC is a rare autosomal recessive disease characterised by prolonged episodes of intrahepatic cholestasis and pruritus alternating with periods of nearly normal liver function, and does not progress to cirrhosis. Since all former approaches to medical treatment of the patients severe pruritus were ineffective, the patient was treated by 3 sessions of albumin dialysis (MARS, Molecular Adsorbents Recirculating System). MARS dialysis decreased serum bilirubin levels by more than 60 % and effectively lowered serum bile acid levels by 45 %. The course of serum parameters was accompanied by a dramatic clinical improvement of the patients symptoms (pruritus, jaundice, fatigue etc.). MARS therapy appeared to shorten the duration of the cholestatic attack.

The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver. Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease. The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP. We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process. The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe. Such selectivity is not observed when free copper is added to N-WNDP. Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper. The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites. Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process. X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1. Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1. The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed.

Wilson disease leads to severe hepatic and neurological pathology resulting from cellular copper overload in the respective tissue. Although the affected gene, ATP7B, has been identified, genetic testing is challenging, time-consuming and expensive. Here we describe the development and use of a novel diagnostic test for four frequent mutations (M769V, W779X, H1069Q and P1134P-fs) found in Germany and many other countries in Europe. The test is based on multiplex polymerase chain reaction and DNA strip technology and was found to be highly sensitive and specific, as well as timely and cost-effective. We conclude that this test is a useful and reliable tool to screen Wilson disease patients and their family members for these mutations and may facilitate diagnosis in this complex disease.

Malignant duodenal obstruction is a common event in patients with advanced biliary tract cancer. Because bypass surgery is accompanied by significant morbidity, self-expandable metallic stents have emerged as a possible alternative for palliation.Twenty patients with biliary tract cancer (7 gallbladder, 13 Klatskin tumors) and duodenal obstruction were treated with metallic stents at a single institution between 1999 and 2001. Survival, morbidity, and stent function were studied prospectively. The ability to eat was assessed by using a scoring system.Stent placement was technically successful in all patients. An additional stent was required in 6 cases (4 occlusions, 2 dislocations). Median survival was 20.5 weeks; there was no treatment-related death. Twenty-eight biliary stent exchanges were performed in 13 (65%) patients. Erosive reflux esophagitis improved in 11 of 12 (92%) cases. After 4 weeks, all 17 surviving patients tolerated soft or solid food, whereas 13 of 17 (77%) tolerated a more solid diet (p0.001, gastric outlet obstruction scoring system). Twelve of 17 (71%) patients gained a median of 1.5 kg of body weight (p = 0.001). The median Karnofsky scale increased from 50% to 60% in 13 of 17 (77%) patients.Self-expandable metallic stents are a safe, efficacious, and minimally invasive treatment option for palliation of patients with duodenal obstruction from biliary tract cancer. Technical complications can be managed endoscopically and the bile duct remains accessible for endoluminal treatment.

Wilson's disease protein (WNDP) is a copper-transporting ATPase essential for normal distribution of copper in human cells. Recent studies demonstrate that copper regulates WNDP through several mechanisms. Six metal-binding sites (MBS) at the N terminus of WNDP are predicted to be involved in copper-dependent regulation of WNDP; however, specific roles of MBS remain poorly understood. To address this issue, we generated WNDP variants with mutations or truncation in the N-terminal region and characterized their functional properties. We show that copper cooperatively stimulates catalytic activity of WNDP and that this effect requires the presence of both MBS5 and MBS6. Mutations of MBS6 or MBS1-5 result in non-cooperative activation of the enzyme by copper, whereas the deletion of MBS1-4 does not abolish cooperativity. Our data further suggest that MBS5 and MBS6 together regulate the affinity of the intramembrane-binding site(s) for copper. Analysis of the copper-dependent stimulation of catalytic phosphorylation demonstrate that the MBS6 and MBS1-5 mutants have a 7-8-fold lower EC50 for copper activation, suggesting that their affinity for copper is increased. This conclusion is confirmed by a markedly decreased inhibition of these mutants by a copper chelator bathocuproine disulphonate. In contrast, deletion of MBS1-4 does not affect the affinity of sites important for catalytic phosphorylation. Rather, the MBS1-4 region appears to control access of copper to the functionally important metal-binding sites. The implications of these findings for intracellular regulation of WNDP are discussed.

Background & Aims: Wilson's disease, a hereditary disorder caused by mutations in the Wilson's disease gene ( ATP7B ), leads to hepatic and/or neurological pathology resulting from cellular copper overload. In vitro studies showed that ATP7B, located in the trans-Golgi network, traffics to a cytoplasmic vesicular compartment in response to increased copper concentration. Mislocalization and failed intracellular trafficking of ATP7B mutants are suggested to be among disease-causing mechanisms; however, the effect of mutations on ATP7B localization in human tissues has not been directly shown. Therefore, we characterized the subcellular localization of normal and mutant ATP7B in human livers and in hepatoma cell lines. Methods: Subcellular distribution of ATP7B in liver tissue from 3 control individuals and 3 Wilson's disease patients harboring a homozygous H1069Q-ATP7B mutation was analyzed by using immunogold electron microscopy. In addition, 14 ATP7B mutants tagged to green fluorescent protein were generated and expressed in HuH-7 and HepG2 cells; intracellular localization of these mutants was characterized by confocal microscopy. Results: In hepatocytes, ATP7B was localized in trans-Golgi vesicles, whereas H1069Q-ATP7B was trapped in the endoplasmic reticulum. Similar results were observed for wild-type ATP7B and H1069Q-ATP7B expressed in hepatoma cells. Most ATP7B proteins harboring missense mutations were distributed similarly to wild-type ATP7B. In contrast, truncated ATP7B mutants showed a diffuse, clustered, cytoplasmic pattern, distinct from the trans-Golgi network or endoplasmic reticulum. Conclusions: These results provide a detailed demonstration of the ATP7B distribution in control and diseased human livers and indicate that several Wilson's disease mutations lead to incorrect localization of ATP7B to distinct cell compartments.

Malignant tumors with high glucose metabolic rates accumulate [18F]-fluorodeoxyglucose (FDG), a positron emitting tracer. The aim of this study was to evaluate FDG positron emission tomography (PET) for detection and staging of human cholangiocarcinoma (CC). Patients with adenocarcinoma of the biliary tree (n = 26), with benign lesions of the bile ducts (n = 8), and 20 control patients underwent FDG-PET (370 MBq [18F]-FDG, Siemens ECAT EXACT HR+). In a blinded fashion, 4 independent experts evaluated the PET scans visually and semiquantitatively using the standardized uptake value and a tumor/non-tumor ratio. All adenocarcinomas and benign lesions (sclerosing cholangitis, bile duct adenoma, Caroli's disease) were histologically proven and imaged by magnetic resonance imaging and endoscopic retrograde cholangioscopy. True-positive PET scans were obtained in 24 of 26 CC and false-negative scans in the other 2 (sensitivity 92.3%). The PET scan was true-negative in 18 of 20 controls and in all 8 benign biliary lesions (specificity 92.9%). Visual and semiquantitative evaluation using tumor/non-tumor ratios were equally accurate (accuracy 92.6%) whereas evaluation by standardized uptake value revealed lower accuracy (P < .05). Regional or hepatoduodenal lymph node metastases were detected with PET in only 2 of 15 cases whereas distant metastases (peritoneal carcinomatosis, pulmonary metastases) were diagnosed in 7 of 10 cases. In conclusion, PET is highly sensitive and specific for the detection and localization of CC. It can be helpful for diagnosis of distant metastases but is not suitable for detection of regional lymph node metastases. (HEPATOLOGY 2001;33:1029-1035.)

Maintenance of isolated retinal Muller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Muller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K + (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Muller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Muller cells using the glutathione-sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate–glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K + , decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended.

Jonas Rosendahl , Heiko Witt , Emmanuelle Masson , Jian-Min Chen , Claude Ferec , Peter Lichtner , Arne Pfeufer , Claudia Ruffert , Peter Bugert , Hana Alg€ ul , Hans B€ odeker , Matthias Bl€ uher , Marco Bruno , Giulia Martina Cavestro , Halina CichozLach , Antonio Farre , Anita Gasiorowska , Andrea Geisz , Elisabetta Goni , Johannes Grothaus , Robert Gr€ utzmann , Stephan Haas , Peter Hegyi , Dominik Huster , Mihai Ioana , Sevastitia Iordache , Grazyna Jurkowska , Peter Kovacs , Monique Derikx . On behalf of the Pan European Working Group Department of Internal Medicine, Neurology and Dermatology, Division of Gastroenterology and Rheumatology, University of Leipzig, Germany Department of Pediatrics, Else Kr€ oner-Fresenius-Zentrum f€ ur Ern€ ahrungsmedizin (EKFZ) & Zentralinstitut f€ ur Ern€ ahrungsund Leben, Germany 3 Etablissement Francais du Sang (EFS) Bretagne and INSERM, U1078, Brest, France, France 4 Institute of Human Genetics, Helmholtz Zentrum M€ unchen, Deutsches Forschungszentrum f€ ur Gesundheit und Umwelt (GmbH), Neuherberg, Germany 5 Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg, Germany 6 II. Medizinische Klinik, Klinikum rechts der Isar der Technischen Universit€ at M€ unchen, Munich, Germany, Germany Kreiskrankenhaus Freiberg, Lehrkrankenhaus der TU Dresden, Klinik f€ ur Innere Medizin, Freiberg, Germany, Germany Department of Internal Medicine, Neurology and Dermatology, Division of Endocrinology, University of Leipzig, Germany, Germany Department of Gastroenterology and Hepatology, Academic Medical Centre, Amsterdam, The Netherlands Vita-Salute San Raffaele University, Division of Gastroenterology and Gastrointestinal Endoscopy, Scientific Institute San Raffa, Italy Department of Gastroenterology Medical University of Lublin, Poland, Poland Dept. of Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, Spain Department of Digestive Tract Diseases, Medical University of Lodz, Lodz, Poland, Poland 14 First Department of Medicine, University of Szeged, Szeged, Hungary, Hungary Dept. of Medicine I, Altona General Hospital, Hamburg, Germany, Germany Department of General, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universit€ at Dresden, Ger, Germany Dept. of Surgical Gastroenterology, Karolinska Institutet CLINTEC, Stockholm, Sweden, Sweden 18 Evangelisches Diakonissenkrankenhaus Leipzig, Akademisches Lehrkrankenhaus der Universit€ at Leipzig, Leipzig, Germany, Germany Molecular and Cellular Biology Department, University of Medicine and Pharmacy Craiova, Romania Department of Internal Medicine and Gastroenterology, University of Medicine and Pharmacy, Craiova, Romania 21 Gastroenterology Department and Internal Medicine, Medical University, Bialystok, Poland, Poland 22 Interdisciplinary Center for Clinical Research Leipzig, University of Leipzig, Germany, Germany Nijmegen, The Netherlands Abstracts / Pancreat

Copper is an essential nutrient required for normal growth and development in many organisms. In humans, the disruption of normal copper absorption and excretion is associated with two severe disorders, known as Menkes disease and Wilson disease, respectively. The consequences of insufficient copper supply that is characteristic of Menkes disease have been largely linked to the inactivation of key metabolic enzymes, although other non-enzymatic processes may also be involved. In contrast, the consequences of copper accumulation in Wilson disease have been generally ascribed to copper-induced radical-mediated damage. Recent studies suggest that the cellular response to copper overload, particularly at the early stages of copper accumulation, involves more specific mechanisms and specific pathways. Genetic and metabolic characterization of animal models of Wilson disease has provided new insights into the pre-symptomatic effects of copper that is accumulated in the liver. The studies have uncovered unexpected links between copper metabolism, cell-cycle machinery, and cholesterol biosynthesis. We discuss these new findings along with the earlier reports on dietary effects of copper. Together these experiments suggest a tight link between lipid and copper metabolism and identify several candidate proteins that may mediate the cross-talk between copper status and lipid metabolism.