An intravenous exposure mouse model for prediction of potential drug-sensitization using reporter antigens popliteal lymph node assay.

Immune-mediated drug hypersensitivity is a particularly concerning health-safety issue among clinicians given its unpredictability and potentially life-threatening effects, especially with exposure to intravenous drugs. Therefore, the development of intravenous drug-exposure models for drug-hazard assessments has garnered increasing interest in recent years. In this study, we used reporter antigens popliteal lymph node assay to investigate the potential value of intravenous exposure to a selected variety of allergenic compounds, including ovalbumin (OVA), concanavalin A (ConA) and diclofenac. The trinitrophenyl (TNP)-specific antibody-forming cells were used to assess the systemic immune responses to a bystander antigen. Mice were subsequently sensitized by TNP-OVA, and then intravenous exposure to one of the selective compounds. As expected, all positive compounds induced significant popliteal lymph node (PLN) proliferation compared with the control. OVA significantly increased Cluster of Differentiation 4 receptors (CD4)⁺ interleukin-4 (IL-4)⁺ T-helper 2 (Th2) cells and, consequently, increased the ratios of IL-4/interferon-γ (IFN-γ) antibody-forming cells (AFCs) in PLNs, while bringing about a dose-dependent increase in immunoglobulin G1 (IgG1) AFCs; these findings indicate that a Th2 hypersensitivity response was induced. A Th2 response was also observed in diclofenac sodium-treated groups, and for ConA, a more mixed Th1/Th2 immune response appeared to be induced. In addition, there was no marked reaction with the negative compound. Together, it seems likely that the intravenous exposure model may be useful for drug-induced systemic hypersensitivity assessments.

Airway responsiveness and bronchoalveolar lavage fluid profiling in individual rats: effects of different ovalbumin exposures.

We studied repeatedly the development of bronchial hyperreactivity (BHR) and bronchoalveolar lavage fluid (BALF) in rats undergoing different modes of ovalbumin exposures. Treatment was two intraperitoneal injections of ovalbumin in Groups 1-3, followed by one ovalbumin aerosolization in Groups 2 and 3, while rats in Group 4 received repeated ovalbumin aerosols after one single intraperitoneal injection. BHR was assessed longitudinally on day 0 (before treatment) and on day 14 (Groups 1 and 2) or 20 (Groups 3 and 4) and cellular influx was estimated from BALF. No BHR or change in BALF cellular profile was detected in Groups 1-3. However, the infiltration of inflammatory cells, associated with BHR (PC(100) 8.9+/-1.3 microg/kg vs. 4.2+/-1.1 microg/kg), was observed in Group 4. The BHR was always associated with increased number of eosinophils in the BALF. The substantial interindividual variability confirmed the need for a technique that permits follow-up of lung responsiveness and BALF profile. This approach evidenced strong associations between the severity of BHR and the eosinophilia.

BCG modulation of anaphylactic antibody response, airwayinflammation and lung hyperreactivity in geneticallyselected mouse strains (Selection IV-A)

The effect of Bacillus Calmette–Gue´rin (BCG) treatment in allergic pulmonary reaction was studied in micegenetically selected accordingly to a High (H-IVA) or Low (L-IVA) antibody responsiveness. Mice wereimmunized with ovalbumin (OVA) or OVA plus BCG. Two days after nasal antigenic challenge, seric IgE andIgG1 anti-OVA, eosinophils in pulmonary tissue, inflammatory cells in bronchoalveolar lavage and the complianceand conductance of respiratory system were evaluated. H-IVA mice were found more susceptible than L-IVA, andBCG was able to inhibit simultaneously the production of IgE, the bronchopulmonary inflammation and bronchialhyperresponsiveness in these genetically selected mice.

Chemical processing as a tool to generate ovalbumin variants with changed stability.

Processing of ovalbumin may result in proteins that differ more than 23 degrees C in denaturation temperature while the structural fold is not significantly affected. This is achieved by 1) conversion of positive residues into negative ones (succinylation); 2) elimination of negative charges (methylation); 3) reducing the proteins hydrophobic exposure (glycosylation); 4) increasing the hydrophobic exposure (lipophilization); or by 5) processing under alkaline conditions and elevated temperature (S-ovalbumin). The effect on the structural fold was investigated using a variety of biochemical and spectroscopic tools. The consequences of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry and by monitoring the tryptophan fluorescence or ellipticity at 222 nm of protein samples dissolved in different concentrations of guanidine-HCl. The impact of the modification on the denaturation temperature scales for all types of modifications with a free energy change of about 1 kJ per mol ovalbumin per Kelvin (or 0.0026 kJ per mol residue per K). The nature of the covalently coupled moiety determines the impact of the modification on the protein thermodynamics. It is suggested that especially for lipophilized protein the water-binding properties are substantially lowered. Processing of globular proteins in a controlled manner offers great opportunities to control a desired functionality, for example, as texturizer in food or medical applications.

The ovalbumin family of serpin proteins.

A protein family, the 'Ov-serpins' has been identified by comparing amino acid sequence, protein characteristics and gene organization. The Ov-serpins would not be recognized as a family based on sequence identity alone. This example suggests that combinations of characteristics may need to be examined to identify family groupings within the serpin superfamily.