Reduction of hepatitis C virus load by H.E.L.P.-LDL apheresis.

The association of HCV with apolipoprotein B containing lipoproteins has been observed and this led to the assumption that the LDL receptor may also serve as a candidate receptor for HCV. H.E.L.P.-LDL apheresis is suggested to be an effective and rapid tool to safely eliminate apolipoprotein B containing lipoproteins. In this pilot study, we have investigated whether H.E.L.P. treatment would reduce HCV load in five patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy (interferon, ribaverin). HCV-RNA was determined by RT-PCR in plasma immediately before the start of apheresis (SA) and after treatment of 2500 mL plasma (AA). H.E.L.P. apheresis led to a mean decrease of 77.3% (16th percentile 36.5%, 84th percentile 89.6%) of HCV-RNA when AA values were compared to SA values. This decline was reproducible during nine treatment procedures, but was not correlated to the decrease in LDL cholesterol. This investigation shows for the first time that HCV load can be reduced by H.E.L.P. apheresis, which is an established and approved therapy for hypercholesterolemia. Even though the efficiency of viral load reduction varied between single procedures and did not correlate to LDL removal, this extracorporeal therapy opens the possibility to treat patients with established immune modulatory and antiviral therapy in the interval between two apheresis procedures.

Effect of heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) apheresis on hepatitis C plasma virus load.

Association of the hepatitis C virus (HCV) with apolipoprotein B containing lipoproteins has been suggested, and this led to the concept that the low-density lipoprotein (LDL) receptor may also serve as a candidate receptor for HCV uptake into the liver. We have investigated whether heparin-induced extracorporeal LDL precipitation (HELP) LDL apheresis treatment reduces HCV plasma load in 6 patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy. HELP apheresis treatment caused an HCV-RNA decrease of 77.3% in mean. This decline was not correlated with LDL-cholesterol reduction. HCV-RNA was retained on the HELP filter as shown for 1 patient. The effect of RNA lowering was only transient due to the high turnover of HCV. However, HELP apheresis may open a window of opportunity for an immune-modulating and antiviral therapy in the interval between two apheresis procedures in patients with high virus load.

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus.

Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03-1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (El/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the El gene product (amino acids 222-336) and the C-terminal part of the E2 protein (amino acids 523-809). Lipoproteins did not bind to recombinant HCV core protein.

Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase-mediated cellular antiviral responses.

The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system. Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR. The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver. ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material. As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique. Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression. The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability.

Differentiation in murine mastocytoma induced by macrophage gangliosides.

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tumor cell division is about 1 microM for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.

Hepatitis B virus core gene mutations which block nucleocapsid envelopment.

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.

Molecular epidemiology of an outbreak of HCV in a hemodialysis unit: direct sequencing of HCV-HVR1 as an appropriate tool for phylogenetic analysis.

Infection with hepatitis C virus (HCV) is still a serious problem in hemodialysis patients, despite screening of blood products for anti-HCV antibodies. The prevalence of HCV in HD patients is between 15% and 30% in Germany. We report the molecular epidemiology of an HCV outbreak in a hemodialysis unit in 1997 is determined. HCV hypervariable region 1 (HVR1) was amplified from serum samples of 19 patients by polymerase chain reaction (PCR) and sequenced directly. In addition, HCV isolates from 3 of these 19 patients were cloned and sequenced. 14 newly infected patients and two patients, who had been infected for several years had very closely related HCV isolates. Unrelated HCV isolates as well as sequences obtained from an HCV outbreak in a plasmapheresis center were found in different, distantly related branches. These findings provide strong evidence for nosocomial transmission of the virus, despite following strict general hygiene precautions. The production of anti-HCV antibody was delayed significantly or seroconversion did not occur at all during the period of observation in 8 out of 14 newly infected HCV RNA positive patients. Close-meshed reverse transcription-polymerase chain reaction (RT-PCR) analyses on apparently non infected patients within hemodialysis units and upon admission of new patients is strongly recommended for the early detection and prevention of outbreaks of HCV.

HIV-Protease inhibitors reduce cell adherence of Candida albicans strains by inhibition of yeast secreted aspartic proteases.

Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients. Secretory aspartic proteases of Candida albicans, which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years. The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated. These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence. As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells. Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect. This inhibition was shown to be concentration dependent. The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases. On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors.

Ratio of serum gamma-GT/ALT rather than ISDR variability is predictive for initial virological response to IFN-alpha in chronic HCV infection.

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-alpha. Because the proportion of patients responding to therapy with sustained or even just with transient elimination of viral RNA is low, several potential prognostic parameters have been evaluated to predict the outcome of the therapy. The present study aimed to prove the validity of a predictive parameter described previously for initial virological response, namely the ratio of serum gamma-glutamyltransferase/alanine transaminase (gamma-GT/ALT) activity in connection with virus genotypes 1a, 1b, and 3a, prospectively and to compare the predictive value of these combined parameters with amino acid variability within the interferon sensitivity determining region (ISDR). The prospective analysis confirmed previous data on the predictive value of the serum gamma-GT/ALT ratio. Concerning ISDR variability, the majority of ISDR sequences obtained from the mostly nonresponding type 1b-infected individuals (23/28) resembled nonmutant types (27/ 28). Isolates from type 3a-infected patients responding to therapy in the majority of cases (13/ 20) exclusively resembled nonmutant types when compared with databank type 3a sequences, but were mutant when compared with the prototype sequence HCV-J. However, the initial virological responsiveness among both type 1b- and type 3a-infected patients did not correlate to ISDR variability. In contrast, virological responsiveness was closely related to serum gamma-GT/ALT ratio. The data are not necessarily contrary to the concept that the number of amino acid exchanges within the ISDR compared with the prototype HCV-J sequence is related to some extent to IFN-alpha sensitivity. The ratio of serum gamma-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter.

Cardiac myocytes of hearts from patients with end-stage dilated cardiomyopathy do not contain Borrelia burgdorferi DNA.

OBJECTIVE: To determine if end-stage dilated cardiomyopathy (DCM) is associated with the presence of Lyme disease causing spirochete Borrelia burgdorferi in the myocardium, we used nested polymerase chain reaction to detect B burgdorferi DNA in myocardial samples from explanted hearts of patients with end-stage DCM. Patients originated from endemic areas for Lyme disease (Bavaria, Lower Saxony, Germany). METHODS AND RESULTS: This was a retrospective study. Polymerase chain reaction was used to detect the specific B burgdorferi recombinant outer surface protein A (OspA) gene in myocardial tissue from 68 patients with end-stage DCM who had undergone heart transplantation. The clinical history of Lyme disease, the presence of Borrelia burgdorferi OspA, and antibodies against OspA in myocardial tissue and serum were investigated. B burgdorferi DNA was not detected in any of the 68 human hearts. Immunoglobulin G antibodies against specific B burgdorferi antigens were observed in 3 (12.5%) of 24 patients. In contrast, 4 hearts from rats experimentally infected with B burgdorferi were all positive for OspA DNA as measured by polymerase chain reaction. CONCLUSION: Our data show that cardiac myocytes of hearts obtained from subjects with end-stage DCM did not contain B burgdorferi DNA as investigated by polymerase chain reaction. However, B burgdorferi shows a high affinity for myocardial tissue as shown by the animal studies, indicating that myocardial infections are nevertheless possible.

Extensive mutagenesis of the hepatitis B virus core gene and mapping of mutations that allow capsid formation.

We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector. The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions. The R-rich 30-amino-acid C-terminal domain was not analyzed. A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen. A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped. Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B. Bottcher, S. A. Wynne, and R. A. Crowther, Nature 386:88-91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop). Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein. Of the 110 mutations, 38 allowed capsid formation in Escherichia coli. They mapped exclusively to nonhelical regions of the proposed fold. The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.

Low density lipoprotein receptor as a candidate receptor for hepatitis C virus.

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 microg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target.

Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations. Eurohep Pathobiology Group.

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up to a factor of 120. The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype adw2) and genotype D (ayw2/3), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion-derived DNA reacted much less efficiently. After identification and elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.4 x 10(9) HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.0 x 10(9) HBV DNA molecules/ml in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organization reference sample.

Characterization of cytostatically active glycosphingolipids isolated from thioglycollate-elicited murine macrophages.

Two acidic glycosphingolipids (gangliosides) derived from mouse macrophage membranes and separated by thin-layer chromatography have a strong cytostatic effect on human and mouse tumor cells. The structure of the two gangliosides, named M phi G1 and M phi G2, was elucidated by application of physicochemical and immunochemical methods. Gas chromatography and mass spectrometry of M phi G1 and M phi G2 classified them as isomeric monosialogangliosides with ceramide moieties composed of sphingosine as the long-chain base, C16 and C18 fatty acids, respectively, and a lacto-tetraose backbone. For M phi G1, additional immunochemical findings led to the proposed structure IV3NeuAc-nLcOse4Cer. The immunochemical reactions of M phi G2 suggest a branched structure for the oligosaccharide moiety.

A membrane-located glycosphingolipid of monocyte/granulocyte lineage cells induces growth arrest and triggers the lytic viral cycle in Epstein-Barr virus genome-positive Burkitt lymphoma lines.

Gangliosides are known to influence cell growth and differentiation. The neolacto series ganglioside IV3NeuAc-nLc4 (2-->3-sialosylparagloboside) is present in members of the monocyte/granulocyte lineage, but is not found in cells that belong to the lymphocyte lineage. In this study we demonstrated that IV3NeuAc-nLc4 inhibits the proliferation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells of the lines Raji and P3HR-1K. IV3NeuAc-nLc4-induced growth inhibition is associated with an increase in G0/G1 phase cells and a reduced expression of CD21 and HLA-DR antigens on Raji cells. These data suggest that IV3NeuAc-nLc4 may affect differentiation of lymphoma cells. Additionally, the increased expression of viral mRNA species which are characteristic for the lytic viral cycle in the non-producer line Raji and the enhanced release of virions from the producer line P3HR-1K demonstrate that IV3NeuAc-nLc4 activates the replication of EBV. Growth inhibition and termination of the viral latency suggest that IV3NeuAc-nLc4 present in monocyte/granulocyte lineage cells may be an effector of the natural defense against EBV persistency and transformation.

Characterization of Borrelia burgdorferi strains in Lyme arthritis.

In the study presented, we investigated whether Lyme arthritis is associated with a particular Borrelia burgdorferi genospecies. Using the PCR technique, in 7/11 samples of synovial fluid of patients with Lyme arthritis a part of the ospA-gene was identified and the strains characterized by sequencing of the amplified DNA. Borrelia burgdorferi sensu stricto was found in 3 patients, B. garinii in 3, and B. afzelii in 1 patient. In conclusion, Lyme arthritis is caused by all 3 human pathogenetic genospecies which are actually known. For clinical practice PCR proved to be a rather insensitive diagnostic method, but may confirm the diagnosis of Lyme arthritis in doubtful cases.

Characterization of unusual escape variants of hepatitis B virus isolated from a hepatitis B surface antigen-negative subject.

Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee's sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.

Autoantibodies with a protective function: polyreactive antibodies against alkaline phosphatase in bacterial infections.

In patients with acute bacterial infections antibodies directed against a particular bacterial antigen were detected. The molecular mass of this bacterial antigen was 50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By comparison of the NH2-terminal amino acid sequence, the 50-kDa antigen was identified as alkaline phosphatase (AP). Affinity-purified antibodies from patient's sera directed against the bacterial AP (anti-alpha) were also shown to react with human and animal AP, which have different structures. Anti-alpha are IgG subtype 3 immunoglobulins, and their light chains are of the kappa type. Upon isoelectric focussing, the anti-alpha formed a scalariform pattern with five to seven bands in the pH range 7-9. The anti-alpha have an opsonic activity and cause a five- to eightfold increase of phagocytosis of gram-positive and gram-negative bacteria. According to their polyreactivity, their sudden rise early in infection, their oligoclonality, as well as their opsonizing properties, they are assumed to be permanently available natural antibodies that take part in early defence mechanisms.

Reinfection in Lyme borreliosis.

Two patients with reinfection of Borrelia burgdorferi are presented. An 11-year-old girl developed recurrent acute peripheral facial palsy at an interval of five years. A 64-year-old woman showed paraesthesia in the leg and effusion in the knee. Three years later, an erythema migrans developed at the thigh. In both patients tick bites, corresponding clinical manifestations, and detection of specific antibodies proved the reinfections. The course of the humoral immune responses showed basic differences between the patients. At the interval between the first and second infection, the specific antibodies of the girl decreased beyond the cut-off level. On the other hand, the titer of specific IgG antibodies of the other patient remained at a constant level. Reasons for the failure of immune protection are discussed.