Search

27 consecutive cases with acute peripheral facial palsy were studied for Lyme borreliosis. In 16 out of 27 children Lyme borreliosis could be diagnosed by detection of specific IgM antibodies in CSF. CSF findings allow a clear distinction according to etiology. All children with facial palsy due to Lyme borreliosis revealed lymphocytic CSF pleocytosis, whereas in cases of unknown etiology CSF was usually normal. Bilateral facial palsy occurred only in children with Lyme borreliosis. All cases with a positive history of tick bite and/or erythema migrans in the head-neck region showed ipsilateral neurological affection suggesting a direct invasion via the affected nerve by Borrelia burgdorferi.

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 tum or cell division is about 1 [_im 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.

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.

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.

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.

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-α. 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 γ-glutamyltransferase/alanine transaminase (γ-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 γ-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 γ-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-α sensitivity. The ratio of serum γ-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter. J. Med. Virol. 58:227–234, 1999. © 1999 Wiley-Liss, Inc.

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.

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 μg/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. J. Med. Virol. 57:223–229, 1999. © 1999 Wiley-Liss, Inc.

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 × 10 9 HBV DNA molecules/ml (range, 2.1 × 10 9 to 3.4 × 10 9 HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 × 10 9 HBV DNA molecules/ml (range, 2.1 × 10 9 to 3.0 × 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.

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.

To determine whether Lyme disease neuropathogenesis may result from infection by a particular segment of the locally extant population of spirochetes, genetic markers of spirochetes found in cerebrospinal fluid (CSF) of 12 pediatric patients were compared with those in spirochetes from 40 vector ticks sampled in the vicinity of their homes. The primary structure of the outer surface protein A served as the marker of variation; a fragment of the corresponding gene was amplified by nested polymerase chain reaction and the products sequenced. Tick-derived variants clustered in seven distinct categories, of which four were present in CSF. One of the CSF variants differed from any found in ticks. Coinfection by different spirochete variants was infrequent in ticks and absent in human samples. Spirochetal neuropathology in children in our study site does not correlate with a particular segment of the tickborne pathogens present in nature.

Cellular immune responses to the HBc or HBe antigen of hepatitis B viruses contribute to the pathogenesis of hepatitis B and to the elimination of the virus. Insufficient cytotoxic immune reactions against the core antigen may be one major reason for viral persistence in the absence of severe clinical symptoms. We attempted to stop viral persistence in the animal model of congenitally infected ducks by injection of recombinant DHBc particles, together with the strong immunostimulator Freund's adjuvant. However, the duck HBc antigen (DHBcAg)-treated ducks did not develop detectable liver disease, nor was the virus persistence affected. The congenitally infected ducks did not contain antibodies against DHBcAg before injection despite continuous production of duck hepatitis B virus, and developed only a weak transient antibody response after injection of recombinant DHBcAg together with Freund's adjuvant. Noninfected ducks developed, in contrast, a strong antibody response to the injected DHBcAg. We conclude that congenitally infected ducks are immunotolerant to DHBcAg and cannot be cured by immunotherapy with exogenous recombinant DHBcAg.

Antibodies directed against the autoantigen p26 were detected in sera from 32 patients with acute Epstein-Barr virus (EBV) infection and clinical symptoms of infectious mononucleosis. P26 has now been identified as the enzyme manganese superoxide dismutase (MnSOD) by comparison of the NH2-terminal amino acid sequence. Antibodies against MnSOD belong to the immunoglobulin class M. They are not detectable in sera of patients with other herpesvirus infections. In the 32 patients investigated, the rise and fall of the autoantibodies coincides with the clinical symptoms. In vitro, the autoantibodies were shown to inhibit the dismutation of superoxide radicals by blocking MnSOD. As presented in the discussion this effect may contribute to the pathogenesis of acute EBV infection.

The hepatitis B virion is a spherical double-shelled particle carrying three surface proteins (large [L], middle [M], and small [S]) in its envelope. All three proteins are translated from a single open reading frame by means of three different in-frame start codons from unspliced mRNAs. This organization defines three protein domains (pre-S1, pre-S2, and S). All three domains together form the L protein, whereas the M protein consists of domains pre-S2 plus S. The L and S proteins are both necessary for virion production, whereas the M protein is dispensable, suggesting an important function of the pre-S1 domain in virion morphogenesis. To investigate this point, we created a series of N-terminal-truncated L mutants and tested their ability to substitute for the wild-type L protein in virion formation. We found that the constructs fell into two classes, (i) N-terminal deletion mutants lacking up to 102 of the 119 amino acids of the pre-S1 domain still allowed virion maturation, showing that the N-terminal 5/6 of the pre-S1 sequence is dispensable for this process. (ii) Mutants lacking 110 or more N-terminal amino acids were unable to substitute for the L protein in virion assembly, although they were stably expressed and secreted as components of subviral 20-nm hepatitis B surface antigen particles. This suggests that a short C-terminal region of pre-S1 is important for virion formation. Like the wild-type L protein, the mutants of the first class were not glycosylated in their pre-S2 domains; however, this site was used for glycosylation in mutants of the second class, similar to that in the M protein. These findings can be related to a model for the function of the L protein in virion maturation.

Lyme borreliosis is a tick-borne infection caused by the spirochete Borrelia burgdorferi, whose discovery in 1982 solved an aetiological mystery involving a variety of dcrmatological and neurological disorders and explained their association with Lyme disease. Lyme borreliosis occurs frequently and is readily treatable with antibiotics. Along with its discovery, however, came the realization that it is difficult to diagnose accurately, especially antibody diagnosis. False-positive antibody results in particular led to gradual widening of the clinical spectrum, and differential diagnosis became increasingly difficult. This prospective, multicentre study presents a systematic description of Lyme borreliosis in childhood, emphasizing epidemiological and clinical issues. Because, predominantly, inpatients were examined, Lyme neuroborreliosis was the focus of the study, with the chief concern being to minimize false-positive results. To this end, we chose to narrow the diagnostic criteria, using the presence of specific antibodies in the cerebrospinal fluid as the determining factor. The epidemiological investigation was focused on the incidence of Lyme neuroborreliosis in childhood in southern Lower Saxony as well as on the prevalence of Lyme neuroborreliosis among acute-inflammatory neurological illnesses in children. The clinical part of the study aimed at establishing criteria for differential diagnosis in addition to the detection of specific antibodies. The detection of specific IgM antibodies using an IgM capture ELISA confirmed the presence of acute Lyme borreliosis. The study examined 208 children with Lyme borreliosis, of whom 169 had Lyme neuroborreliosis, from mid-1986 until the end of 1989. The yearly incidence of Lyme neuroborreliosis in Lower Saxony was 5.8 cases/100,000 children aged 1 to 13. The manifestation index was 0.16, or one case of Lyme neuroborreliosis per 620 infected children, compared with the presence of specific antibodies against B. burgdorferi for children in the same age group and region. Both the seasonal distribution of Lyme borreliosis, which peaked in summer and autumn, as well as the information about when the tick bites took place point to an incubation period of a few weeks. The most frequent manifestation of Lyme neuroborreliosis in childhood was acute peripheral facial palsy, found in 55% of all cases (n = 93). Lyme borreliosis proved to be the most frequently verifiable cause of acute peripheral facial palsy in children, causing every second case of this disorder in summer and autumn. Bilateral facial palsy was, without exception, found to be caused by Lyme borreliosis; thus it can be considered a specific neurological sign of this infection. The second most common manifestation of Lyme neuroborreliosis in childhood was aseptic meningitis (27.2%, n = 46). Lyme borreliosis was the third most common cause of aseptic meningitis in childhood (11.8%). Meningoradiculoneuritis with peripheral nerve involvement (Bannwarth syndrome) was diagnosed in only 3.6% of the children (n = 6), although this is the most common symptom of Lyme neuroborreliosis in adult patients. The head and neck region proved to be the predominant site of tick bites in children; adults experience most bites on their extremities. The difference in the site of the infection, as well as the short duration of the illness in children and the early treatment, may partially explain the profound differences in the clinical spectrum of Lyme neuroborreliosis in children and adults. Nearly all cases with a positive history of tick bite and/or erythema migrans in the head and neck region showed ipsilateral facial palsy suggesting a direct invasion via the affected nerve by B. burgdorferi. Inflammatory changes in the cerebrospinal fluid along with the presence of specific antibodies are the conditio sine qua non for a diagnosis of Lyme neuroborreliosis. The presence of IgM antibodies in the cerebrospinal fluid has proved to be the most reliable diagnostic criterion, and is responsible for the early diagnosis of acute Lyme neuroborreliosis in children. The IgM capture ELISA facilitated the simple and reliable detection of intrathecal antibody synthesis which could be successfully demonstrated in three-quarters of the cases with Lyme neuroborreliosis. Specific IgG testing was negative in most cases because of the relatively short duration of the illness in children, and is therefore of less importance here than in the diagnosis of cases of Lyme neuroborreliosis in adults. Acute Epstein-Barr and varicella-zoster viral infections both caused false-positive findings in the IgM capture ELISA. High dose intravenous penicillin G proved to be highly effective in paediatric cases of Lyme neuroborreliosis. Although the rate of spontaneous remission may be very high, it is not possible to predict on a patient-by patient basis just when this will occur. Thus consistent antibiotic therapy is indicated.

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03-1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19-1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-beta lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-beta lipoprotein. In 3 sera no significant precipitation could be observed.

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

The nucleotide sequence of a 1.6-kb clone containing the gene for outer surface protein A (OspA) of a German strain (GO2) of Borrelia burgdorferi was determined. The deduced amino acid sequence showed a homology of 82% to the OspA molecules from three other B. burgdorferi strains. The best-conserved region was recognized at the 36-amino-terminal amino acids of OspA. OspB could not be identified in the strain investigated, probably because the nucleotide sequence of the ospAB operon prevented expression of the OspB gene.

Antibodies against gangliosides isolated from small tumour and nervous tissue specimens can be reliably detected in serum samples by the ganglioside electrotransfer technique without the need for previous purification steps. After separation by high performance thin layer chromatography gangliosides are transferred from silica gel plates to hydrophobic polyvinylidene difluoride membranes. These membranes are highly suitable for immuno-staining. The use of a 15-slit device allows simulataneous testing of up to 15 serum samples. Samples of serum from 39 patients with clear-cell carcinoma of the kidney, mammary carcinoma, ovarian carcinoma and neurological disorder together with samples from healthy controls were tested for anti-ganglioside antibodies from various tissues.

Sera from 118 women of 33 to over 90 years of age, with or without a history of cervical squamous-cell carcinoma, were examined for the presence of antibodies to HPV-6b, HPV-16 and HPV-18, L1, L2, E4, and E7 gene products by the use of bacterially derived beta-Gal fusion proteins and Western-blot analysis. Among the cervical cancer patients, 29/46 (63.0%) were positive for antibodies to E4 and/or E7 of HPV-16 and/or E7 of HPV-18. In contrast, only 2 of 31 (6.5%) non-genital cancer patients and 4 of 41 (9.8%) healthy individuals were antibody-positive for HPV-16 E4 or E7, while antibodies to the homologous proteins of HPV-18 could not be detected. Prevalence rates of antibodies to the HPV-16/18 late proteins were 25/46 (54.3%) in the cervical carcinoma group, 13/31 (41.9%) among women with non-genital cancer types, and 18/41 (43.9%) among normal, healthy individuals. Antibodies to HPV-6b late gene products ranged between 6.5% and 12.2% in the different patient groups. Antibodies to HPV-6b E4 and E7 were detected only once. By studying an additional control group of 207 women with a different age distribution, age-dependence of antibodies to HPV gene products could be ruled out. Whereas antibodies to late proteins may indicate that, regardless of clinical stage, HPV infections are wide-spread among the female population, the striking difference between the prevalence rates of antibodies to early proteins of HPV-16 and HPV-18 among cervical cancer patients and controls (p less than 0.001) supports the idea of the involvement of these virus types in carcinogenesis of the cervix.

Open reading frames of human papillomaviruses were expressed in Escherichia coli as β-galactosidase fusion proteins. These bacterially derived papillomaviral gene products were used to examine sera from 67 women (63 healthy subjects, 4 patients with genital carcinoma) for antibodies to papillomavirus type-16 antigens (El, E2, E4, E5, E6, E7, L1, L2) and the L2 proteins of HPV-6b and HPV-18 by Western-blot analysis. The serologic data were compared with cytological findings classified according to Papanicolaou and with nucleic acid hybridization data from cervical smears of the same individuals. Twenty-three of the normal individuals showed antibodies exclusively directed against L2 gene products; whereas in the sera from the four genital cancer patients, antibodies to the early gene products E4 and/or E7 could be detected. In one case these antibodies were found to be combined with antibodies to L2 of HPV-16 and −18 and in another case with those to El and E2 of HPV-16. In none of the sera examined could antibodies to L1, E5 or E6 be identified. Three of the antibody positive normal women were found to be also positive for HPV-16/18 DNA, while all of the 40 seronegative women were HPV-16/18 DNA negative. These data indicate that serology may be a valuable means to study the epidemiology of genital human papillomavirus infection.

We have developed a modified ELISA for the detection of anti-Borrelia burgdorferi (Bb) antibodies based on a peroxidase enzyme labelled antigen (ELAT). Microtiter plates were coated with antigen of Bb. The immunoglobulins of the serum samples were bound to the antigen and specific antibodies were detected by an enzyme labelled antigen. The test principle facilitates the recognition of specific antibodies in different collectives of human and animal sera. We performed epidemiological studies with the ELAT on 231 sera from mothers in maternity wards (9.5% positive), 219 patient sera sent to the Bb routine diagnostics (15% positive) and 230 sera from forestry workers (21.3% positive). We further investigated sera from red deer from South Lower Saxony which remained 55% Bb-antibody positive; deer were 37% and fallow deer were 29% positive.

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

The diagnostical significance of the large hepatitis B surface protein with its preS1 attachment site and of anti-preS antibodies are not yet well known. We investigated the epitope of the preS1 attachment site to see whether it is a marker of viremia and whether antibodies against it occur in convalescents and vaccinees. For comparison, sera were also tested for the presence and relative amount of a preS2 epitope. The epitopes were detected by binding to specific monoclonal antibodies (mAb MA18/7 for the preS1 epitope and mAb Q19/10 for the preS2 epitope) at the solid phase of a sandwich enzyme-linked immunosorbent assay. Antibody against the preS1 epitope was detected by inhibition of binding to mAb MA18/7. This mAb inhibits attachment of preS1 antigen to hepatocytes and reacts with a subtype-independent sequential epitope at the surface of hepatitis B virus between amino acid 29-36. This preS1 epitope occurs in most hepatitis B surface antigen (HBsAg) carriers, irrespective of viremia. Free preS2 epitope Q19/10 is present in samples with more than 8 micrograms/ml total HBsAg and it is masked in sera with less HBsAg. Antibodies which compete with mAb MA18/7 for its viral preS1 epitope occur in one third of HBsAg carriers who were negative for hepatitis B e antigen. It also occurs in one third of convalescents and in most good responders to plasma-derived vaccines.

Spirochete diversity in acrodermatitis chronica atrophicans lesions in a closely defined central European site was compared to that in the local vector population, in human erythema migrans lesions, and in cerebrospinal fluid by amplifying and sequencing a segment of the gene of outer surface protein A directly from sampled tissues. Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi acutely infect human skin and invade internal tissues. Only B. afzelii, however, is associated with acrodermatitis chronica atrophicans lesions, persisting chronically where the skin has atrophied.

During the course of acute Epstein-Barr virus (EBV) infection, there is a rise of oxygen radical production. As a consequence, the production of the oxygen radical scavenger manganese superoxide dismutase (MnSOD) is increased. Patients with acute EBV infections regularly develop autoantibodies against MnSOD that are able to inhibit the enzyme activity in vitro. To elucidate the origin of the autoantibodies, the epitopes on MnSOD were determined. The entire sequence of MnSOD was synthesized as overlapping pentadecapeptides, which were scanned for their reactivity with sera of patients with acute EBV infections. Sera as well as affinity-purified anti-MnSOD antibodies reacted with the peptides p(no15) (amino acids 47-61) and p(no30) (amino acids 122-136) lying in crucial parts of the MnSOD tetramer. The two main epitopes p(no15) and p(no30) showed sequence homologies with EBV-encoded proteins. Reactivity of affinity-purified antibodies with a peptide of the homologous BGLF4 points to a molecular mimicry causing the occurrence of anti-MnSOD antibodies. Anti-MnSOD antibodies were able to block the protective effects of MnSOD in a model for oxidative damage produced by xanthine/xanthine oxidase in EAhy926 endothelial cells. Thus, these autoantibodies may contribute in vivo to the clinical symptoms by accumulation of toxic oxygen radicals.

In most sera of hepatitis C virus (HCV)-infected patients beta-lipoproteins are bound to HCV RNA-carrying material, most often simultaneously with immunoglobulins (IgG, IgM) and sometimes additionally with high-density lipoproteins, forming complexes of low density (1.04-1.06 g/ml). To separate HCV particles from bound material, we tried to destroy the lipoprotein enzymatically by incubating HCV-positive human sera with lipoprotein lipase derived from Pseudomonas spp. (LPL-Ps). After this treatment, titers of HCV RNA in human sera (determined by a simple semiquantitative reverse transcription-PCR assay) were strongly reduced, regardless of whether primers of the NTR or NS5 region were used. Inactivation of HCV RNA could be inhibited by the addition of RNAguard to the serum-enzyme mixture. The lytic effect of the LPL-Ps preparation could be inhibited by tetrahydrolipstatin. Hence LPL-Ps seems to disrupt the HCV particle structure, including the putative core of the virus, and makes HCV RNA sensitive for RNase present in the reaction mixture. HBV was not destroyed by LPL-Ps. Porcine pancreatic lipase had no effect on HCV. The implications of these observations for the structure and biology of HCV and for its stability and inactivation in human sera are discussed.

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.

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.

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.

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.

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. (Am Heart J 1999;138:269-72.)

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.

The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3'-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories.

The preS domain at the N-terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post-translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.

Having returned from a holiday in Southeast Europe, a 30-year-old German woman developed acute hepatitis. Hepatitis A virus (HAV) infection was diagnosed serologically. During the course of the infection, hemolysis was found. IgM antibodies against triosephosphate isomerase (IgM anti-TPI) were detected in the patient's serum from the acute phase of the HAV infection. Affinity purified IgM anti-TPI from the serum reduced the enzyme activity in vitro and caused an increased51 Cr release from erythrocytes. IgM anti-TPI is assumed to be one of the causative agents of hemolysis in HAV infection.

Lyme borreliosis is a common infectious disorder in children. The daily life and play routines in particular make them more likely than adults to be bitten by ticks and thus more likely to be infected by Borrelia burgdorferi. Lyme borreliosis as a clinical entity was first recognized in children suffering from arthritis (1). In the meantime, research in this field has mainly been focused on the disease in adults. Epidemiological data about the true incidence of Lyme borreliosis in childhood are still sparce. The whole clinical spectrum of Lyme borreliosis described in adults is also found in children, but age-specific differences in the relative frequency of certain manifestations, in the course of the disease as well as in diagnostic pecularities are evident (2–9).

Heterogeneities in the density of hepatitis C virus RNA-carrying material (HCV-RNA-CM) found in human sera (1.03-1.20 g/cm3) are attributed to the binding of low-density lipoproteins and/or of IgG. In some sera HCV-RNA-CM seems to be nearly totally bound to beta-lipoproteins and cannot be precipitated by anti-IgG (gamma); in others more than 95% of HCV-RNA-CM is bound to IgG and cannot be precipitated by anti-beta-lipoprotein. Furthermore, there are sera from which HCV-RNA-CM can be completely be precipitated by either anti-beta-lipoprotein or anti-IgG (gamma), pointing to a binding of the two serum proteins to the same HCV-RNA-CM. There are other sera from which HCV-RNA-CM can be partially precipitated by the one or the other antiserum, leaving behind fractions, which are bound to beta-lipoprotein or to IgG. HCV-RNA-CM cannot be precipitated from some sera either by anti-beta-lipoprotein or by anti-IgG (gamma).

The prevalence and time course of the occurrence of antibodies to the hepatitis B virus polymerase (anti-HBpol) were investigated in acutely and in chronically HBV-infected individuals by using recombinant HBpol protein for Western blot analysis. One group consisted of 19 patients who were acutely infected and recovered completely. Five of these patients (26%, 69 serum samples examined) exhibited anti-HBpol. Among those anti-HBpol positive patients, recovery from the disease was combined with a complete loss of this antibody. In contrast, in a second group of 15 individuals who developed chronic hepatitis B, 13 (87%, 102 serum samples examined) had anti-HBpol during the acute phase of the disease. The difference between the anti-HBpol prevalence rates of the two patient groups is statistically significant (Exact Fisher test, P < .002), implying that the occurrence of anti-HBpol may be indicative of a potential chronic course of hepatitis B. Remarkably, anti-HBpol was found in one case of a clinically suspected hepatitis B in which no other serological HBV parameters were found. This serum sample was positive in HBV PCR, supporting a possible diagnostic value of anti-HBpol. © 1993 Wiley-Liss, Inc.

In half of the sera from patients with acute bacterial infections and 15% of the sera from patients with acute viral infections glycoproteins were found that form a scalariform pattern in the cationic range upon isoelectric focusing. The cationic glycoproteins appeared with the clinical illness. After subsidence of the symptoms they disappeared within 4 to 6 weeks. The proteins were identified as immunoglobulin G (IgG) by determination of the NH2-terminal amino acid sequence. Remarkably, these IgG only contained light chains of the kappa type with high proportions of carbohydrates. Both, N-glycosidic- and O-glycosidic-bound glycans were present. The glycosylated light chains may render the cationic IgG multireactive. Thus, it may be part of an early nonspecific immune defense mechanism.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Background 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. Materials and methods 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). Results 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. Conclusions 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.

Summary. DNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector A gt 1 1. After infection of the Escherichia coli strain Y 1089, the plaques of recombinant phages were screened with a B. burgdorferi antiserum (human) for fusion proteins containing borrelia antigens. A positive clone produced a hybrid protein (p200) of c. 200 Kda. The corresponding native borrelia protein (p97) was identified as having an M, of 97 Kda. To localise protein p97 in the B. burgdorferi cell, immunoelectronmicroscopy and a Western blot of isolated flagella were used. Antibodies directed against proteins p200 and p97 recognised epitopes associated with the flagella.

Investigations of immunoblots using Borrelia burgdorferi antigen demonstrated that a band, migrating faster than the bromophenol blue front in sodium dodecyl sulfate-gel electrophoresis, reacted strongly with sera containing anti-Borrelia burgdorferi antibodies preferentially of the IgG class. Extraction of this antigenic component and chemical analyses showed that the substance was composed mainly of fatty acids and carbohydrates. Typical structures of classical lipooolysaccharides such as 3-deoxy-D-manno-2-octulosonic acid, hydroxy fatty acids or lipid A could not be detected.

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with β-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.

The DNA fragment of the human parvovirus B19, with 715 nucleotides between nucleotide positions 3141-3856 was expressed in Escherichia coli as a beta-galactosidase fusion protein. The plasmid vector pSS20d used for this purpose permits cleavage of the viral gene product from the beta-galactosidase moiety by collagenase. After purification by p-aminophenyl-beta-D-thiogalactoside-sepharose and superose, a soluble protein with a molecular mass of 28 kDa was isolated. It represents a common part of the viral capsid proteins VP1 and VP2. This bacterially derived parvoviral gene product can be used for detection of anti-B19 antibodies in human sera.

In the course of infectious mononucleosis, IgM antibodies are formed against 2 proteins present in nucleated and non-nucleated vertebrate cells. Antibodies were found in sera of all patients suffering from acute Epstein-Barr virus infection. In 40% of the cases these antibodies are monoclonal. Persons with former Epstein-Barr virus infection were negative. The antibodies against the 2 proteins were first detected in Raji cells with an IgM-specific immunofluorescence test. The proteins were demonstrated in extracts of different cells and tissues by immunoblot technique. The molecular weight of the proteins measured in SDS polyacrylamide gel electrophoresis was 26 kd and 29 kd, respectively. Their presence in the cells does not depend on the presence of the Epstein-Barr virus genome. The relevance of the new findings concerning diagnostics as well as pathogenetic aspects remains to be established.