Preparation of immunoblot test stripes from a Rubella virus-like particles dye crystal complex as antigen.

Stably transfected Chinese hamster ovary (CHO24S) cells were the source for Rubella virus-like particles (RVLP) containing all structural proteins (E1, E2, C and their dimers). RVLP are secreted from the CHO24S cells into the medium and the time-point for collecting the medium with the highest yield of >100 kDa proteins (with 17 mg protein from 10 ml cell culture supernatant) was after 2 days of incubation. Different methods for RVLP isolation from the cell culture supernatants were assessed by SDS-PAGE and Western blotting (using sera positive or negative for Rubella virus (RV)-specific antibodies or an anti-E1 monoclonal antibody). A combination of membrane filtration with a rapid, novel gradient ultracentrifugation step (using Coomassie brilliant blue G crystals as adsorbens for RVLP that facilitated virus isolation) was the most suitable technique. 132 RV-positive human sera (RV IgG > 20 IU/ml by commercial ELISA) were tested by our "self made" immunoblot test stripes (using RVLP adsorbed to dye crystals as antigen) for the presence or absence of antibodies specific for RV structural proteins. 57.6% of these sera had antibodies against E1, E2 and C, 31% against E1 and C, and 1.5% against E1 only, whereas 3.8% had no RV specific antibodies and only 6.0% were equivocal which demonstrated that these "self made" test stripes can reliably differentiate RV antibody specificities.

Epidemiology of rubella infections in Austria: important lessons to be learned.

In order to ascertain the epidemiology of rubella infections in Austria, a seroepidemiological study was performed. Data collected from 115 cases diagnosed at the Institute of Hygiene and Social Medicine of the University of Innsbruck during 2001 were evaluated. The results indicate this infection can no longer be categorised as a paediatric disease (mean age, 18.5 years), and several other findings were particularly striking: (i) 47% of the patients had elevated C-reactive protein levels and 50% had increased anti-streptolysin O titres; (ii) only a few patients complained of fever, while symptoms such as rash and lymphadenopathy, which are also associated with several other viral infections, including HIV, were found frequently; and (iii) the 115 rubella cases detected in the 1-year study period represented an incidence of >13 per 100,000 population. This high incidence of infection underlines the need for further improvement of diagnostic tests and more successful vaccine strategies.

Correlation of low-density lipoprotein modification by myeloperoxidase with hypochlorous acid formation.

Myeloperoxidase is an enzyme in phagocytes which catalyzes several redox reactions. A major product is hypochlorous acid which appears to be important in inflammatory processes such as atherosclerosis. The aim of this study was to investigate whether the kinetics of low-density lipoprotein modification by the myeloperoxidase/hydrogen peroxide/chloride system in vitro conform to the established kinetics of hypochlorous acid formation and to compare the results with known in vivo data. The absorbance at 234 nm was applied to study the kinetics of the modification of low-density lipoprotein. Variation of the concentration of low-density lipoprotein, hydrogen peroxide, and chloride, respectively, had a biphasic effect on the maximal rate of low-density lipoprotein modification. Increasing the substrates up to certain threshold levels resulted in increased modification, however, further increases caused inhibition of low-density lipoprotein modification. The inhibitory effect of higher low-density lipoprotein concentrations might be relevant, since these concentrations occur in the human aortic intima. Furthermore, a positive correlation was found between the maximal rate of low-density lipoprotein modification and the acidity of the medium. In summary, low-density lipoprotein modification is affected by the myeloperoxidase/hydrogen peroxide/chloride system in a similar manner to hypochlorous acid production. We conclude that myeloperoxidase, which has been detected in atherosclerotic lesions, is able to modify low-density lipoprotein into the form which is taken up by macrophages in an uncontrolled manner.

Chondroitin 4-sulphate exhibits inhibitory effect during Cu2+-mediated LDL oxidation.

Chondroitin 4-sulphate (C4S), a basic component of the extracellular matrix of the artery wall, inhibited copper-induced low density lipoprotein (LDL) oxidation by prolonging the lag time and reducing the rate of propagation. LDL oxidation was assessed by monitoring conjugated dienes and low level chemiluminescence. A possible initial key reaction in LDL oxidation, the reduction of copper(II) to copper(I) by LDL, was decreased in the presence of C4S. Moreover, C4S protected tryptophan residues of Apo-B-100 in the early stage of LDL oxidation and during the subsequent propagation phase. The presence of the sulphate group in position 4 of N-acetylgalactosaminyl residues of C4S is crucial for protective activity. In fact, the structurally related chondroitin 6-sulphate, also present in tissues, had no effect on LDL oxidation. These data suggest that C4S may contribute to protect LDL against oxidation in arterial intima.

Formation of N-formylkynurenine suggests the involvement of apolipoprotein B-100 centered tryptophan radicals in the initiation of LDL lipid peroxidation.

Tryptophan oxidation products were determined in pronase E digests of apo B of delipidated, native and Cu2+ oxidized LDL using a sensitive and specific HPLC method. Oxidized LDL contained N-formylkynurenine, kynurenine and tryptamine but no oxindolylalanine and 5-hydroxytryptophan. N-Formylkynurenine increased from an initial value of 0.21 to 1.67 mol/mol apo B within 5 h. Apo B of native LDL also contained kynurenine (0.80 mol/mol) and tryptamine (0.13 mol/ mol). The results support the assumption that oxidation of Trp residues in apo B is an early event and possibly an elementary reaction involved in initiating LDL oxidation.

Effect of stobadine on Cu(++)-mediated oxidation of low-density lipoprotein.

The pyridoindole derivative stobadine [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido(4,3b) indole] has been described as a drug with antihypoxic and antiarrhythmic cardioprotective properties. The antioxidative properties of this compound were studied during Cu(++)-mediated low-density lipoprotein (LDL) oxidation. Stobadine (concentration 0-5 microM) prolonged the lag phase (in min produced by one molecule antioxidant per LDL particle) as measured by diene formation more effectively than did ascorbate, trolox, or alpha-tocopherol. It also has the ability to decrease the rate of diene formation during the propagation phase very efficiently. Diene formation, Trp destruction, and alpha-tocopherol consumption were measured in the presence and absence of stobadine. Stobadine (10 microM) did not influence tocopherol consumption during oxidation and the Trp fluorescence quenching of Cu++ was not influenced by this compound. From these results, as well as polarographic measurements, we conclude that the antioxidative effect of stobadine is not simply a result of Cu(++)-ion complexation. In contrast to ascorbate, this compound is stable in the presence of Cu++. Stobadine inhibits the oxidation of LDL-Trp residues very efficiently via its radical scavenging properties, and may even have the ability to reduce Trp radicals to tryptophan. The concentration of stobadine used for LDL oxidation was in the range found in plasma (stobadine given p.o. in human and rats results in plasma concentrations between 0.2-3.9 microM.

Early destruction of tryptophan residues of apolipoprotein B is a vitamin E-independent process during copper-mediated oxidation of LDL.

The decrease of the tryptophan fluorescence (Ex/Em = 282/331 nm) was used to monitor the kinetics of copper-mediated LDL oxidation. Cu2+ causes a concentration-dependent quenching of the LDL Trp-fluorescence, the maximum of about 22% suggests that 8-9 Trp residues (out of a total of 37) are accessible for Cu2+ ions. Decomposition of LDL tryptophan commences immediately after addition of Cu2+ and proceeds in two stages with quite different rates. At a molar ratio of Cu2+/LDL = 33:1 the LDL particle looses 1 Trp every 13.5 min in the initial slow phase and every 4.1 min in the subsequent rapid The second, stage temporarily coincides with the propagating lipid peroxidation. In the initial phase loss of Trp proceeds with a constant rate for up to 200 min depending on the copper concentration. Whereas lipid peroxidation accelerates after consumption of vitamin E, rate of Trp loss does not increase. Loading of LDL with vitamin E has also no effect on the initial rate of Trp loss. During the initial phase a loss of one Trp residue/LDL is accompanied by the loss of two alpha-tocopherols and the generation of two conjugated lipid hydroperoxides. The results suggest Trp residues play a role in initiating the lipid peroxidation process in the LDL particle. In such kinetic studies, precautions must be taken to avoid photodecomposition of LDL-Trp. The LDL vitamin E fluorescence (Ex/Em = 290/323 nm) does not interfere with the Trp fluorescence even at high concentrations.