Sven G. Gehrke

Pro-hepcidin: expression and cell specific localisation in the liver and its regulation in hereditary haemochromatosis, chronic renal insufficiency, and renal anaemia

Background and aims: The hepatic peptide hormone hepcidin, which has recently been isolated from human plasma and urine, is thought to be a central regulator of iron homeostasis. We investigated the presence and cellular localisation of hepcidin in the liver and developed a non-invasive assay to analyse its regulation in patients with hereditary haemochromatosis (HH), chronic renal insufficiency (CRI), and renal anaemia (RA). Methods: Expression and localisation of hepcidin was shown by reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, and immunofluorescence in human and guinea pig liver. Serum concentrations were determined in various groups of patients using a sensitive enzyme linked immunosorbent assay (ELISA). Results: Western blot analysis with region specific antibodies identified a ~10 kDa peptide corresponding to the apparent molecular mass of pro-hepcidin. Localisation studies revealed that pro-hepcidin is expressed at the basolateral membrane domain of hepatocytes and is also present in blood. We developed a stable sensitive ELISA for detection and determination of pro-hepcidin in human serum. Mean pro-hepcidin level in human serum of healthy volunteers was 106.2 ng/ml. Enhanced levels of pro-hepcidin (148.1 ng/ml) were found in patients with CRI but normal haemoglobin values, indicating that the kidneys may metabolise and/or eliminate the circulating hormone. In contrast, concentrations of pro-hepcidin were significantly decreased in patients with HH (70.2 ng/ml) and also in patients with RA (115.0 ng/ml) compared with the CRI group. Conclusions: From the detection of pro-hepcidin in human serum, we conclude that the prohormone may be involved in the regulation of iron metabolism in HH. Decreased pro-hepcidin levels could play an important role in the pathogenesis of HH.

Angiotensin II Stimulates Matrix Metalloproteinase Secretion in Human Vascular Smooth Muscle Cells via Nuclear Factor-κB and Activator Protein 1 in a Redox-Sensitive Manner

The renin-angiotensin system contributes to atherogenesis. Matrix metalloproteinases (MMP) are thought to participate in plaque destabilization through degradation of extracellular matrix. This study tested whether angiotensin II (ANG II) induces MMP in human vascular smooth muscle cells (SMC). ANG II induced expression of MMP-1, -3, and -9, but not of MMP-2 in SMC. The expression of MMP-1, a key enzyme for collagen degradation, was studied in detail. SMC stimulated with ANG II concentration-dependently released enzymatically active MMP-1. The ANG II type 1 receptor antagonists losartan and candesartan blocked ANG-II-induced MMP-1 release. Inhibition experiments with actinomycin D suggest ANG-II-induced MMP-1 mRNA regulation at the transcriptional level. Decoy oligodeoxynucleotides against nuclear factor-ĸB and activator protein 1 inhibited MMP-1 secretion, demonstrating participation of these transcription factors in MMP-1 transcription. Stimulation of MMP-1 by ANG II depended on cyclooxygenase 2. The antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine, the flavin protein inhibitor diphenylene iodonium, and the NADP(H) oxidase inhibitor apocynin blocked MMP-1 release, suggesting a redox-sensitive mechanism involving NADP(H) oxidase. The reactive oxygen species (ROS) donor 2,3-dimethoxy-1,4-naphthoquinone induced MMP-1 secretion and enhanced ANG-II-stimulated MMP-1 expression. These findings indicate that ROS may increase their own production by activation of NADP(H) oxidase. The capability of ANG II to induce functionally active MMP in human SMC may contribute to the altered plaque composition seen in complicated stages of atherosclerosis.

Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin

Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe−/− mice, an Hfe−/− mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe−/− mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe−/− mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.

Hemochromatosis and transferrin receptor gene polymorphisms in chronic hepatitis C: impact on iron status, liver injury and HCV genotype.

Mild iron overload in chronic hepatitis C is associated with liver fibrosis, hepatitis C virus (HCV) genotype 1b infection, and an impaired response to interferon therapy. In this study we evaluated whether polymorphisms in the hemochromatosis gene HFE and the transferrin receptor gene TFR1 are associated with these typical findings. The study considered 246 HCV-infected patients and 200 blood donors as controls, in which C282Y, H63D, and S65C mutations (HFE) and the S142G polymorphism (TFR1) were detected. HCV genotype, serum ferritin levels, stainable intrahepatic iron, and grade of fibrosis according to the METAVIR score (F0–F4) were determined. In HCV-infected patients, heterozygosity for the C282Y mutation in HFE was significantly associated with elevated serum ferritin levels, stainable liver iron, and advanced fibrosis or cirrhosis (F2–F4). By multivariate logistic regression analysis the odds ratio for the development of advanced fibrosis or cirrhosis (F2–F4) was 2.5 for HCV-infected patients carrying a heterozygous C282Y mutation and 4.8 for HCV-infected patients with C282Y/H63D and C282Y/S65C compound heterozygosity. Heterozygosity for the C282Y mutation in HFE contributes to iron accumulation and fibrosis progression in chronic hepatitis C.

Iron Stores Modulate Hepatic Hepcidin Expression by an HFE-Independent Pathway

Background/Aims: In HFE-related hereditary hemochromatosis an inappropriately low hepatic expression of the iron-regulatory peptide hepcidin (encoded by HAMP) has been suggested to cause iron overload. The aim of the present study was to evaluate whether the hepatic expression of HAMP in relation to iron stores requires HFE or might involve other important iron-related genes including HJV (encoding hemojuvelin) and TFR2 (encoding transferrin receptor-2). Methods: Using quantitative RT-PCR, the iron-dependent hepatic expression patterns of HAMP, HJV, and TFR2 were evaluated in human and murine HFE-related hemochromatosis. Results: The overall level of hepatic HAMP expression in human and murine HFE-related hemochromatosis is impaired but can still be modulated by iron stores. Moreover, we demonstrate an HFE-independent correlation between the expression of HAMP and TFR2 in mouse and human livers. On the other hand, a strong correlation between the hepatic expression of HAMP and HJV was only found in hemochromatosis patients and Hfe-deficient mice. Conclusion: The central pathogenetic step in HFE-related hemochromatosis is an impaired basal expression of HAMP rather than a lack of HAMP upregulation in response to iron stores. An HFE-independent pathway that seems to involve TFR2 and HJV can regulate HAMP expression under conditions of iron overload.

First clinical trial of a newly developed capsule endoscope with panoramic side view for small bowel: A pilot study

Capsule endoscopy is the first‐line diagnostic technique for the small bowel. However, the inability to visualize the duodenal papilla is an inherent limitation of this method. In the present study, we evaluated feasibility of a newly developed CapsoCam SV1 capsule.

SLA/LP/tRNP(ser)Sec antigen in autoimmune hepatitis: Identification of the native protein in human hepatic cell extract

A diagnostic subgroup of AIH type 1 is characterized by specific serum antibodies against soluble liver protein. The respective autoantigen was named SLA/LP/tRNP((Ser)Sec), after three homologous recombinant polypeptides were isolated from expression gene libraries. We analyzed human cultured liver cells for the human homologue of recombinant SLA/LP/tRNP((Ser)Sec) by antigen purification. In addition, a monoclonal antibody was generated against recombinant SLA-p35, a truncated recombinant SLA-reactive polypeptide. With a positive patient serum, immune affinity chromatography was performed on the 52 kD-SLA main antigenic determinant pre-enriched by ion exchange chromatography. By mass spectrometry, the 52 kD-SLA/LP/tRNP ((Ser)Sec) autoantigen was unambiguously identified in the purification product. The identity of the recombinant SLA-p35 and its human homologue was further confirmed by a specific signal of the anti SLA-p35 monoclonal antibody with purified human SLA/LP/tRNP((Ser)Sec). The 48 kD-SLA species frequently comigrating in SLA-immunoblotting however was not identified by either approach. We conclude that the native counterpart of recombinant tRNP((Ser)(Sec)) indeed is detectable with a molecular weight of 52 kD in soluble liver extract of human cells as the major antigenic component of SLA/LP/tRNP((Ser)Sec).

Evidence for a critical role of ceruloplasmin oxidase activity in iron metabolism of Wilson disease gene knockout mice

Background and Aim:  Wilson disease is a genetic disorder associated with copper overload due to mutations within the ATP7B gene. Although copper and iron metabolism are closely linked, the influence of mutations of the ATP7B gene on iron homeostasis is unknown. Therefore, the present study was carried out to elucidate iron metabolism in Atp7b(−/−) mice, an animal model of Wilson disease.

Hämochromatose und Morbus Wilson

Zum ThemaIn der vorliegenden Übersichtsarbeit wird das derzeitige Wissen über die beiden autosomal rezessiv vererbten Erkrankungen Hämochromatose und M. Wilson zusammengefaßt und jeweils über deren Ätiologie, Pathogenese, Klinik, Diagnostik, Therapie und Prognose referiert. Während die Prävalenz der Hämochromatose, die hierzulande zu den häufigsten Erbkrankheiten zählt, vergleichsweise hoch ist (Heterozygote 1:10 bis 1:20, Homozygote 1:200 bis 1:400), ist der M. Wilson seltener (Heterozygote ca. 1:90, Homozygote ca. 1:30.000).Je früher die Diagnose beider Erkrankungen gestellt wird, um so günstiger ist unter geeigneter Behandlung die Prognose für die Lebenserwartung, die an die der Normalbevölkerung heranreicht. Bei Hämochromatose sind konsequent durchgeführte Aderlässe die Therapie der Wahl, bei M. Wilson die Therapie mit Chelatbildnern, im Extremfall die Lebertransplantation.In der Diagnostik spielt die molekulargenetische Testung eine zunehmend wichtige Rolle, die allerdings bei M. Wilson sehr schwierig und noch sehr aufwendig ist, weswegen sie bei dieser Erkrankung als Standarddiagnostik gegenwärtig nicht zur Verfügung steht.

The Detergent Effect of Mesalazine Interferes with Phosphatidylcholine Binding to Mucin 2

Objectives: Therapeutically applied delayed-release phosphatidylcholine (PC) revealed mucosa protection and clinical improvement of ulcerative colitis. However, a recent trial with simultaneous application of delayed-release PC and mesalazine showed lack of efficacy. It is hypothesized that mesalazine acts as detergent to prohibit PC integration into mucus as target compartment, thus preventing topical mucus protection. Methods: In vitro PC-binding studies with mucin 2 and intestinally differentiated CaCo2 cells as well as outcome analysis of a therapeutic trial with delayed-release PC and additional mesalazine. Results: Choline-containing phospholipids, in particular PC, bind to mucin 2 as main scaffold protein of intestinal mucus to establish a hydrophobic barrier towards microbiota in the intestinal lumen. PC also binds to the apical surface of polarized CaCo2 cells with membrane-anchored mucin 3. Mesalazine removes mucin-bound PC and, thus, reduces transepithelial resistance. A post hoc analysis of patients from a previous multicenter phase IIB trial with delayed-release PC revealed that those without mesalazine showed a PC dose-dependent outcome with regard to achievement of partial and complete remission (p < 0.05 for 1.6 and 3.2 g PC daily) whereas those treated simultaneously with mesalazine showed no PC dose dependency. Conclusion: Mesalazine solubilizes PC and, thus, prevents the protective action of therapeutically applied delayed-release PC within mucus.