Measuring the Kinetics of Neutral Pair Evaporation from Cluster Ions of Ionic Liquid in the Drift Region of a Differential Mobility Analyzer.

Singly charged clusters [C+A-]nC+ or [C+A-]nA- of two salts [C+A-] are produced by electrospray ionization of alcohol solutions of the ionic liquids 1-ethyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate (EMI-FAP) and 1,2-dimethyl-3-propylimidazolium-methide (DMPI-Me). The rate of neutral pair evaporation into [C+A-] + [C+A-]n-1C+ or [C+A-]n-1A- is studied in atmospheric pressure as a function of temperature T for the positive trimer ion (n = 2) of DMPI-Me and the negative trimer ion of EMI-FAP. The trimer is separated from all other electrosprayed ions in a first differential mobility analyzer (DMA1) and then transferred through a cooled tube to a second DMA whose drift gas is kept at a controlled temperature (25 °C < T < 100 °C). Singular characteristics of the DMA are a residence time τ of ∼0.1 to 1 ms, with essentially uniform temperature and τ. The decomposition occurring within DMA2 results in a complex mobility spectrum associated with dimer product ions, with apparent mobilities intermediate between those of the dimer and the trimer, depending on the product of the reaction rate k and τ. A theoretical expression yielding k from the shape of the collected mobility spectrum is obtained by accounting for the deterministic reactive, convective, and diffusive evolutions of the parent and product ions within DMA2. Observed and predicted mobility spectra agree well, yielding the reaction rate k with little ambiguity. Activation energies near 1 eV are determined for both trimer ions. Paradoxically, the evaporation process substantially heats up the cluster ion product. The theory developed enables measuring decay times much smaller and much larger than the residence time in the DMA.

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli.

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.

A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization.

Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.

Expression and immunological evaluation of the Escherichia coli-derived hepatitis C virus envelope E1 protein.

Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1(340), amino acids 192-340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1-encoding peptides. From this, 51.1% of human sera were found to react with E1(340). High-level antibodies against E1(340) were obtained in rabbits and mice when immunized. These antibodies had a similar peptide-recognition pattern to that described previously for human sera. The most reactive region was located at the N-terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed-type hypersensitivity assay. It revealed the induction of a CD4+ T-cell-mediated response by this protein. This E1(340) protein and the animal-derived anti-E1 sera are immunological tools that could aid in the monitoring and development of anti-HCV therapies.

Lack of significant bronchial reactivity to inhaled normal saline in subjects with a positive methacholine challenge test.

Patients with symptoms suggestive of asthma often have normal resting pulmonary function. In these patients, a determination of airway responsiveness by bronchial challenge is useful in demonstrating bronchial hyperreactivity (BHR), a defining feature of asthma. In the methacholine (Mch) challenge, it is recommended that following a baseline measurement of FEV1, the patient inhale the normal saline (NS) diluent and FEV1 be repeated to assess for nonspecific BHR to NS. It is also recommended that post-NS inhalation FEV1 should be used as the control value from which decrement in FEV1 is compared following Mch challenge. Mch testing was performed in 44 patients with symptoms suggestive of asthma (cough, chest tightness, dyspnea) and normal resting pulmonary function. Baseline spirometry was obtained and repeated after inhalation of NS and after five breaths each of Mch at the following concentrations: 0.025 mg/ml, 0.25 mg/ml, 2.5 mg/ml, 10 mg/ml, and 25 mg/ml. The procedure was terminated when FEV1 decreased to at least 80% of the post-NS value or if the maximal concentration of Mch had been reached. The post-NS FEV1 value was > or = 91% of the pre-NS value in all the subjects range 91-105%). Using the post-NS FEV1 as the recommended control value, 20 patients (45%) had a positive Mch challenge and 24 patients (55%) had a negative Mch challenge. Had we used the pre-NS FEV1 as a control value, only 2 patients would have been reclassified, and when these 2 cases are carefully examined, there would have been no significant change in the clinical interpretation of the MCh test.(ABSTRACT TRUNCATED AT 250 WORDS)