Martin Volkmann

In vitro experimental infection of primary human hepatocytes with hepatitis B virus

BACKGROUND/AIMS Studies on the interaction of hepatitis B virus (HBV) with its host cell require a suitable tissue culture system. This study used primary adult hepatocytes from healthy human liver tissue to establish productive infection in vitro. METHODS Hepatocytes were inoculated overnight with HBV. Production of viral proteins was assessed by radioimmunoassay and by [35S]methionine labeling, and production of viral DNA was assessed by Southern blotting and endogenous polymerase assay. RESULTS Secretion of high levels of hepatitis B surface antigen (HBsAg) and low levels of hepatitis B virus e antigen (HBeAg) into the medium was detectable 6 days after infection and reached maximum values after 12 days. Metabolic labeling showed production of viral proteins to be a result of de novo synthesis. The appearance of single-stranded HBV DNA in the cytoplasm of infected cells, typically present in immature cores, indicated viral replication. HBV DNA containing particles possessing an active viral DNA polymerase could be immunoprecipitated from the medium 12 days after infection. An antiserum specific for the preS1 region of the viral envelope was capable to block infection. Presence of dimethyl sulfoxide in the medium greatly improved the yield of viral proteins. CONCLUSIONS Primary adult human liver cells are competent for infection with HBV.

Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia

The blockade of the platelet integrin glycoprotein (GP) IIb/IIIa has proved to be an effective antiplatelet therapy. Profound thrombocytopenia has repeatedly been described as an adverse effect in patients treated with GP IIb/IIIa inhibitors, but its mechanism has not been elucidated yet. With use of flow cytometry, the activation status of platelets was monitored in 26 patients presenting with acute myocardial infarction who were treated with the GP IIb/IIIa inhibitor abciximab alone or in combination with the fibrinolytic agent reteplase. Fibrinogen and PAC-1 (a GP IIb/IIIa activation-specific monoclonal antibody) binding, as well as P-selectin expression on unstimulated platelets were constant in 25 patients throughout a follow-up of 7 days. In 1 patient (D.F.), the percentage of platelet-binding fibrinogen increased from 2.2% to 17.8%, for PAC-1 from 2.8% to 13.2%, and for P-selectin expression from 10.2% to 58.3% 10 minutes after the start of treatment. Furthermore, D.F. had a decrease in single platelet count in ethylenediaminetetraacetic acid-, citrate-, and heparin-anticoagulated and native blood. Blood films revealed platelet aggregates. In vitro testing of D.F.s blood 2 and 4 weeks after initial admission demonstrated a reinduction of fibrinogen and PAC-1 binding to platelets, an increase of P-selectin expression, and formation of platelet aggregates following exposition of platelets to abciximab in vitro. In summary, this report describes the induction of platelet activation by a GP IIb/IIIa inhibitor in vivo and reinduction in vitro in direct association with thrombocytopenia. Platelet activation by GP IIb/IIIa inhibitors may be one potential mechanism for GP IIb/IIIa inhibitor-induced thrombocytopenia.

Evaluation of the serum-free light chain test in untreated patients with AL amyloidosis

The diagnostic value of serum electrophoresis and urinary light chain excretion in AL amyloidosis is poor due to the usually low amount of monoclonal protein. This study evaluates immunoglobulin light chains in the serum of a large series of untreated patients with systemic AL amyloidosis, and proves that free light chain levels reflect disease severity. We evaluated the Serum Free Light Chain (FLC) test in a series of 133 untreated patients with systemic AL amyloidosis. The FLC test detected the monoclonal gammopathy in 87% compared with 92% for immunofixation of serum and urine in combination. However, both tests proved complementary. The FLC test was also a valuable tool in patients with advanced renal failure in spite of uninvolved light chain retention. Higher FLC levels were associated with higher bone marrow plasmocytosis, poorer Karnofsky index and heart involvement, and therefore reflected disease severity.

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.

Cerebrospinal fluid tau levels in Alzheimer's disease are elevated when compared with vascular dementia but do not correlate with measures of cerebral atrophy

Increased tau levels are a well-established finding in Alzheimers disease (AD). In contrast, the potential value of tau levels in the differential diagnosis of AD, vascular dementia (VD) and major depression warrants further investigation. The potential impact of psychotropic medication also needs to be established. We investigated cerebrospinal fluid (CSF) tau protein concentrations in 88 patients with AD, 23 patients with VD, 25 patients with major depression and 17 age-paralleled controls without cognitive impairment with respect to important clinical variables, type and dosage of psychotropic medication and cerebral changes as assessed by magnetic resonance imaging (MRI). The AD patients showed significantly elevated tau levels compared with patients with VD or major depression and controls. Tau levels obtained in the VD group were intermediate, with significant differences from both AD patients and patients with major depression and controls. Within the AD group, no significant correlation between tau levels, severity of dementia, age, duration of disease, type and dosage of psychotropic medication or MRI volumetric changes arose. A subgroup of AD patients without increased tau levels was characterized by a significantly larger percentage of patients with presenile onset.

Levels of total tau and tau protein phosphorylated at threonine 181 in patients with incipient and manifest Alzheimer's disease

Cerebrospinal fluid tau protein levels are considered to be a promising marker for Alzheimers disease (AD) and may facilitate early detection. Using the newly developed INNOTEST Phospho-Tau((181P)) kit examination of total tau and tau protein phosphorylated at threonine 181 (phospho-tau 181) revealed significantly (P<0.05) higher values in both patients with incipient and manifest AD than in controls. In patients with vascular dementia, phospho-tau 181 levels were not different from controls but were significantly lower than in patients with manifest AD. These findings suggest that total and phosphorylated tau protein may facilitate early detection and differential diagnosis of AD.

Loss of CD95 expression is linked to most but not all p53 mutants in European hepatocellular carcinoma

Abstract. Experimental data have shown p53-dependent CD95 induction to be associated with increased levels of apoptosis after cytostatic treatment in hepatoma cells. A study of Japanese hepatocellular carcinoma (HCC) has reported an inverse correlation between CD95 and p53 expression. To examine the interaction of p53 and CD95 in tumors we investigated which alterations in p53 can be linked to loss of CD95 expression in European HCC. In 39 tumors we analyzed CD95 by immunohistochemistry and assessed the correlation between the findings of the p53 status as determined by immunohistochemistry and single-strand conformation polymorphism with polymerase chain reaction sequencing. In 10 of 14 tumors with evidence of p53 aberration there was also loss of CD95 expression, compared to 6 of 25 samples with apparent wild-type p53 (P<0.01). Three tumors with p53 mutations but sustained CD95 expression showed single base substitutions mapping to a narrow region of 20 codons in p53. A significant correlation with differentiation status of the tumor was found for the p53 aberration but not for CD95 expression. This is the first study to link loss of CD95 expression to specific p53 alterations in HCC. Functional p53 appears to be a major factor for CD95 expression in hepatocytes, the loss of which could contribute to chemoresistance and possibly immune evasion in hepatocellular carcinoma. Sustained CD95 expression in tumors with certain p53 aberrations may indicate functional heterogeneity of p53 mutants.

Increased T-Helper 2 Cytokines in Bile From Patients With IgG4-Related Cholangitis Disrupt the Tight Junction–Associated Biliary Epithelial Cell Barrier

BACKGROUND & AIMS IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. METHODS We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)-associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na(+)/Cl(-) permeability, and 4-kDa dextran flux analyses. RESULTS Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. CONCLUSIONS Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

The impact of elevated serum IgG4 levels in patients with primary sclerosing cholangitis.

BACKGROUND Elevated IgG4 levels have been reported among patients with primary sclerosing cholangitis. Epidemiological data has only been provided from tertiary centres. AIMS To investigate the prevalence of elevated IgG4 levels and to compare prognosis between patients with and without elevated IgG4 levels in serum in two European cohorts of patients with primary sclerosing cholangitis. METHODS Serum IgG4-levels were measured in a consecutive series of patients from Berlin, and retrospectively collected in a population-based cohort from Sweden (total N=345). Coxs proportional hazard analysis was used to calculate relative risks for liver-related death or liver transplantation and cholangiocarcinoma. RESULTS Elevated IgG4 values were demonstrated in 10% of patients. A previous history of pancreatitis, combined intra- and extrahepatic biliary involvement and jaundice were independently associated with elevated IgG4 in multivariate analysis. IgG4 status was not associated with an increased risk for the combined endpoint liver-related death or liver transplantation or cholangiocarcinoma. CONCLUSION The prevalence of elevated IgG4 values among European patients with primary sclerosing cholangitis is similar to what previously has been reported from the United States. Elevated IgG4 was not associated with an increased risk of liver transplantation or liver-related death or cholangiocarcinoma.

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).