Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

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.

Manganese superoxide dismutase as a target of autoantibodies in acute Epstein-Barr virus infection.

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.

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.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

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.

Identification of an endoflagellar associated protein in Borrelia burgdorferi.

DNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector lambda gt11. After infection of the Escherichia coli strain Y1089, the plaques of recombinant phages were screened with a B. burgdorferi antiserum (human) for fusion proteins containing borrelia antigen.s A positive clone produced a hybrid protein (p200) of c. 200 Kda. The corresponding native borrelia protein (p97) was identified as having an Mr 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.

Capture and RT-PCR of hepatitis C virus RNA with safety primers.

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.

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product.

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.

Occurrence of human papillomavirus DNA types 16 and 18 (HPV-16/18) in cervical smears as compared to cytological findings.

Cervical smears from 2336 women were examined for the presence of HPV-16/18 by dot-blot hybridization using 32P-labeled HPV-16/18 DNA under high stringency conditions. The hybridization data were compared with cytological findings classified according to Papanicolaou. The ages of the patients ranged from under 20 to over 70 years. Ninety-eight (4.4%) of the 2237 cytologically normal cervical samples (Pap I and II) were HPV-16/18 positive. Thirteen out of 32 (40.6%) samples showing signs of mild and moderate dysplasia (Pap IIID) were found to be HPV-16/18 positive. In 5 out of 7 (71.4%) samples from women with severe dysplasia or carcinoma in situ (Pap IV) and in 9 out of 25 (32.1%) samples from patients with invasive cervical carcinoma (Pap V) HPV-16/18 DNA was detected. Thirty-two smears were from women with severe unspecific cervical inflammation (Pap III). Two (6.2%) out of them were HPV-16/18 positive. Normal smears showed an apparent age-dependent pattern of HPV-16/18 positivity with a peak prevalence of 10.6% among women younger than 20 years old. The majority of premalignant lesions was detected among women younger than 40 years old; whereas all invasive lesions were from women older than 39 years. Compared to the HPV-16/18 prevalence rate in normal smears, abnormal smears harbored HPV-16/18 DNA approximately 9 times more frequently. This finding supports the hypothesis that HPV-16/18 may be involved in the development of cervical cancer.

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.

Epidemiology and clinical manifestations of Lyme borreliosis in childhood. A prospective multicentre study with special regard to neuroborreliosis.

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 dermatological 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.(ABSTRACT TRUNCATED AT 400 WORDS)

[Rubella vaccination with "Cendehill" and "RA 27/3". Reinfection of vaccinated and naturally immune persons compared with the infection rate of susceptible persons within a 3-resp. 5-year observation period (author's transl)]. / Feldversuch zur Wertbemessung von Rötelnimpfstoffen. Röteln-Impfung mit "Cendehill" und "RA 27/3", die. Reinfektionshäufigkeit der geimpften und natürlich immunen Personen im Vergleich mit der Infektionshäufigkeit empfänglicher personen innerhalb einer 3- bzw. 5jährigen Beobachtungszeit

Many investigators in Europe and the USA have usually found high rates of serum conversion after vaccination with the rubella vaccines Cendehill and RA 27/3, but the resistance of vaccinated individuals against superinfection without disease induced by wild virus strains under experimental or natural conditions seems to be very low; e.g. up to 80% of the vaccinees reacted with increasing titers of hemagglutination inhibiting antibodies after super-infection with a wild virus in a special epidemiological situation. In our two studies performed under natural conditions of infection including an observation period of three years resp. five years after vaccination a more favourable picture evolved, perhaps reflecting a different epidemiological situation, a different susceptibility or a less virulence of wild virus in our region: Three years after vaccination of 14 years old girls with Cendehill and RA 27/3 in a double blind trial a significant increase in the titers of hemagglutination inhibiting antibodies was observed after vaccination with Cendehill by the subcutaneous route in only 1,16% (1/86) and in 1,18% (1/85) after vaccination with RA 27/3 by the same route. In the same observation period the infection rate in a control group not protected by natural immunity or by vaccination was 54,72% (87/159) and in a second control group, protected by natural immunity 1,82% (3/165)...

Live attenuated versus killed virus vaccines.

The discussion of the problems of production and use of killed and live virus vaccines shows that the theoretical, basic concepts of the alternative vaccination procedures are equivalent in regard to the ideals of calculable potency and safety. If one compares the present stages of development, then it becomes evident that the advantages of live viral vaccination are due to greater potency. However, killed virus vaccines are safer. Nevertheless, these differences are not without limits. In some vaccinating procedures, the live virus vaccine is the safer of the two.

Mapping a region of the large envelope protein required for hepatitis B virion maturation.

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.

Electron-microscopic identification of infectious particles of lymphocytic choriomeningitis.

LCM virus, strain WE--grown on L cells--and labeled with 3H-uridine was centrifuged to equilibrium in a sucrose density gradient and examined in fractions for infectivity, incorporated radioactivity, and electron-microscopic features. The peak of infectivity is congruent with the one of radioactivity (density = 1.17 g/ml). LCM virus specificity of the radioactive peak was proved by precipitation of the radioactivity with anti-LCM virus antiserum. The peak fractions showed an abundance of 106 +/- 14 nm (1s) particles. They could be agglutinated with specific anti-LCM virus antiserum but not with antiserum directed against the histocompatibility (H-2) antigens of L cells.

Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells.

Vaccinia virus specific cytotoxicity against infected target cells was observed in vitro. Spleen lymphocytes from normal and immunized mice of the inbred strains C3H and DBA/2 were incubated with vaccinia virus-infected and non-infected 51 Cr-labeled mastocytoma P-815-X2 cells and L-929 fibroblasts, which were used as targets. Cytotoxic lymphocytes could be isolated from the mice as early as 2 days after infection with vaccinia virus. The highest cytotoxic effect was obtained with lymphocytes taken 6 days after infection. The degree of lysis was correlated with the ratio of immune lymphocytes to target cells. Specific blocking of target cell lysis resulted after addition of anti-vaccinia antibody from different sources. The effector cells could be characterized as T cells by elimination of macrophages and B cells. Target cell killing was only possible in a syngeneic system; allogeneic infected target cells were not lysed significantly.

Standardized detection of hepatitis B surface antigen: determination of its serum concentration in weight units per volume.

The concentration of HBsAg in serum was determined in arbitrary units by quantitative immunoelectrophoresis and in units of optical density by UV-photometry of purified antigen. The HBsAg was purified from serum by gelchromatography and subsequently by isopycnic centrifugation in cesium chloride. The ratio of the immunoelectrophoretically measured concentration to the units of optical density was found to be constant in six different samples with both subtypes - ad and ay - and varying concentration. The concentration of protein in three purified HBsAg samples was determined after acid hydrolysis by automatic aminoacid analysis. The specific extinction was E1 280 mg/ml = 4,5 +/- 0,3. Thus the arbitrary units of immunoelectrophoresis were converted to concentration of HBsAg specific protein, expressed as mug/ml. In 540 Ausria positive serum samples, mainly from the beginning of an acute hepatitis, 10-40 mug/ml was the most frequent range of concentration. About 4% of the sera contained more than 100 mug/ml, about 20% contained between 0,005 and 0,5 mug/ml and were positive only in RIA. 10 mug/ml correlated with titers in complement fixation of 1:32/64, in counterimmunoelectrophoresis of 1:8 and immuno-diffusion of 1:2. The use of standards with a defined concentration of HBsAg would allow a better control of the sensitivity in qualitative tests and of reproducibility in quantitation.

IgM autoantibodies against two cellular antigens always appear in acute Epstein-Barr virus infection.

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.

[Infectious mononucleosis: hemolysis by autoantibodies against triosephosphate isomerase]. / Infektiöse Mononukleose: Hämolyse durch Autoantikörper gegen Triosephosphat-Isomerase.

Generalized adenopathy and splenomegaly developed in an 18-year-old youth after a severe tonsillitis followed by headache, tiredness and weight loss for several weeks. Infectious mononucleosis (acute Epstein-Barr virus infection) was confirmed by the demonstration of virus-specific antibodies. A reticulocytosis (24%), decreased haptoglobin concentration (0.6 mg/dl) and increased lactate dehydrogenase activity (657 U/l) indicated marked haemolysis. The bone marrow showed increased erythropoiesis with abnormal maturation. Antibodies against triosephosphate isomerase and against blood group marker "i" were demonstrated in the patient's serum. Antibodies against triosephosphate isomerase from the patient's serum were purified by affinity-chromatography. They strongly reacted with the patient's erythrocytes and under complement activation induced an increased 51Cr liberation from marked erythrocytes. No corresponding effect of anti-i-antibodies was noted at 37 degrees C. With the fall in antibodies against triosephosphate isomerase the haemolysis receded and the patient became free of symptoms after 7 weeks.