[Hepatitis B reactivation following immunosuppressive therapy and screening adherence: a neglected problem?]. / Hepatitis-B-Reaktivierung unter Chemotherapie und Screening-Adhärenz: ein unterschätztes Problem?

BACKGROUND: Current guidelines of the "Centers for Disease Control and Prevention [CDC]" recommend routine screening for Hepatitis B before cytotoxic or immunosuppressive therapies are initiated. The national German guideline "Prophylaxis, diagnosis and therapy of hepatitis B virus infection" is in line with the CDC recommendations and underscores general HBV screening before immunosuppression is induced. However, screening adherence and acceptance of these guidelines vary in different oncological specialities. To assess the HBV screening adherence a retrospective study was performed. PATIENTS AND METHODS: Data of 140 patients were analyzed retrospectively. 37 case-records did not meet inclusion criteria. Patients diagnosed with breast-cancer (n = 43) and Hodgkin's disease (n = 14) requiring chemotherapy were included, as well as patients receiving allogenic stem cell transplantation (SCTx) therapy (n = 22) or transarterial chemoembolization (TACE) therapy of the liver (n = 24). All included case-records were reviewed regarding HBV and HCV serology. RESULTS: In the TACE group three patients were screened for HBsAg. Four patients with breast cancer and five patients in the Hodgkin disease group were screened for HBsAg. In contrast, screening adherence was 100 % in the group of patients receiving allogenic stem cell transplantation therapy (n = 22). CONCLUSION: Apart from patients with allogenic stem cell transplantation, only some patients receiving immunosuppressive therapies had been screened for HBV infection. Our data indicate that standardized checklists may improve HBV screening previous to immunosuppressive therapies. These clinical structures have led to an almost optimal screening adherence in the high-risk group of allogenic SCTx patients.

[kappa-light chain amyloidosis of the liver, a rare cause of liver enzyme elevation]. / Leichtkettenamyloidose der Leber vom kappa-Typ. Seltene Ursache einer cholestatischen Leberenzymkonstellation.

HISTORY AND ADMISSION FINDINGS: A 69-year-old man was admitted to the department of gastroenterology having for months had persistently elevated liver enzymes after discontinuing systemic antimycotic therapy. He reported loosing five kilogram of body weight in the past six months. No macroscopic or microscopic abnormalities had been found on esophago-gastroduodenoscopy. INVESTIGATIONS: Congo-red staining of the liver biopsy revealed massive sinusoidal amyloidosis of the liver. Immunoelectrophoresis of the urine and serum, as well as bone marrow biopsy, ruled out multiple myeloma or Waldenström's disease. Immunohistochemical staining identified the amyloid protein as a IgG kappa light chain (KLC). The free light chain (FLC) test confirmed KLC monoclonal gammopathy with an abnormal free kappa to lambda chain (KLLC) ratio. TREATMENT AND COURSE: Systemic KLC amyloidosis in this patient older than 65 years was given chemotherapy with melaphalan and dexamethasone (M-Dex). After three courses of M-Dex the renal clearance deteriorated and the serum N-terminal probrain natriuretic peptide (T-proBNP) had increased. COURSE: The patient was included in a phase II clinical trial which evaluates the use of bortezomib in patients with amyloidosis. Normalization of the free KLLC ratio and the NT-proBNP level will serve as important prognostic indicators. CONCLUSION: KLC amyloidosis is a rare cause of elevated liver enzymes. The nonspecific symptoms often delay the diagnosis. FLC testing is a helpful tool in identifying monoclonal gammopathies, even when immunoelectrophoretic tests are normals.

Inhibition of duck hepatitis B virus replication by intrahepatic expression of an antiviral resistance gene.

Resistance genes coding for inhibitors of hepadnaviral replication, such as ribozymes, antisense RNA, and dominant negative mutants have been shown to be effective in transfected hepatoma cells. In vivo studies, however, are not available to date. Here we expanded the use of the duck hepatitis B virus (DHBV) model for studying antiviral resistance genes in vivo. Animals were experimentally infected by intravenous injection of DHBV-positive serum in ovo. The use of recombinant human adenovirus type 5 and avian adenovirus CELO for gene transfer was evaluated. Adenovirus type 5 transduced more than 95% and CELO less than 1% of embryonic hepatocytes in vivo. Adenovirus type 5 interfered with DHBV replication (viral cross-talk), but this effect was moderate and did not preclude analysis of specific antiviral effects. Thus adenoviral transfer of a dominant negative mutant prior to DHBV infection (intracellular immunization) yielded 100-fold suppression of viral replication compared to the green fluorescent protein marker gene. Neither gene was toxic. These data demonstrate that a prototype anthepadnaviral resistance gene is functional in vivo. Duck embryos represent a useful model for evaluating gene therapeutic strategies in vivo without the need for large scale preparations of gene delivery vehicles.

Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.

Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

[Chronic diarrhea--value of function tests in diagnosis]. / Chronische Diarrhöe--Stellenwert der Funktionstests in der Diagnostik.

The D-xylose-adsorption test yields information about small intestinal absorptive function. To investigate ileal absorptive function the Schilling test is performed. The standard diagnostic test for hypolactasia is the oral lactose tolerance test with lactose breath hydrogen testing. Various radioactive and nonradioactive breath tests have been proposed as noninvasive tests for small intestinal bacterial overgrowth. Intubation tests for pancreatic function testing are time-honored and direct, but limited by several technical difficulties. Measurement of pancreatic enzymes in stool has received a high degree of acceptance.

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo.

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.

Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis.

Dominant negative (DN) mutants of the hepadnaviral core protein are potent inhibitors of viral replication. We have previously shown that fusion of sequences derived from the duck hepatitis B virus (DHBV) polymerase (Pol), DHBV small surface protein (S), bacterial beta-galactosidase (lacZ), or green fluorescent protein (GFP) to the carboxy terminus of the DHBV core protein yields DN mutants that inhibit viral replication at the posttranslational level. To elucidate the mechanism(s) of their antiviral action, we analyzed the effect of the DN mutants on RNA pregenome packaging and nucleocapsid assembly. Core-Pol and core-S, but not core-lacZ or core-GFP, markedly interfered with RNA pregenome packaging. Nucleocapsid formation was not affected by any of the mutants. The DN core-GFP fusion protein formed mixed particles with wild-type core protein in the cytoplasm of cotransfected cells and interfered with reverse transcription of the viral pregenome. A subpopulation of chimeric nucleocapsids, however, was shown to overcome the block in DNA synthesis and produce mature viral DNA. Thus, at least 2 steps within the viral life cycle can be targeted by DN DHBV core proteins: 1) packaging of the viral pregenome; and 2) reverse transcription within mixed particles. The fact that some mixed particles retain replication competence demonstrates a high structural flexibility of nucleocapsids and indicates a possible mechanism of viral escape.

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations.

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.

Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo.

Efficient DNA delivery is a prerequisite for the successful implementation of molecular antiviral strategies against chronic viral hepatitis and gene therapy in general. The cationic polymer polyethylenimine (PEI) has recently been explored as a gene transfer vector in various cell types in vitro and in vivo. In this study, we evaluated a linear PEI derivative (lPEI) as a vector for gene and oligodeoxynucleotide transfer into hepatocytes in vitro and in vivo. A simple protocol was developed that allowed transfection of up to 50% of primary hepatocytes in vitro. In addition, fluorescent oligodeoxynucleotides were efficiently delivered to the liver in vivo after intravenous injection into Pekin ducks. Thus, lPEI mediates highly efficient gene and oligodeoxynucleotide transfer into primary hepatocytes and is potentially useful for DNA delivery in vivo.

Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection.

The aim of this study was to characterize the interferon induced intracellular signals in duck hepatocytes and to investigate the effects of duck interferon on virus replication in duck hepatitis B virus (DHBV) infected ducks. Interestingly, duck interferon was found to activate intracellular signal transduction pathways similar to those of its mammalian counterparts. An interferon stimulated gel shift activity like that of gene factor 3 is induced, as well as serum inducible element binding factors homologous to serum inducible factor A (SIF-A), SIF-B and SIF-C. Duck interferon induced signal transducer and activator of transcription activation is not inhibited by DHBV infection of hepatocytes. DHBV infected ducks treated for 10 days with recombinant duck interferon show a decrease in viral DNA in hepatocytes, and in many cases disappearance of viraemia. These findings confirm the usefulness of the DHBV infection model for the study of human hepatitis B virus infection.

Antisense therapy of hepatitis B virus infection.

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30-40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5'-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.