Cross-reactive immune responses in mice after genetic vaccination with cDNA encoding hantavirus nucleocapsid proteins.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in about 150,000 individuals in Eurasia, and several hundred cases of hantavirus pulmonary syndrome (HPS) on the American continent annually. There is consequently a need for rapid diagnostics and effective prevention of hantaviral infections. In this study we have performed DNA-vaccination of mice with full-length genes encoding the immunogenic nucleocapsid protein (NP) of Puumala (PUUV), Seoul (SEOV) and Sin Nombre virus (SNV). The antibody reactivity towards the NPs, and deleted or truncated variants thereof, were studied to localise and investigate the major polyclonal B-cell epitopes. Our findings clearly show that the antibody reactivity in each immunised mouse is unique, not only in a quantitative respect (titers) but also in cross-reactivity and most likely also in the epitope specificity. Our experimental data in combination with B-cell prediction software indicate that strong homologous virus species specific and cross-reactive epitopes are located around amino acid residue 40 in the nucleocapsid proteins.

The nitrogen mustard melphalan activates mitogen-activated phosphorylated kinases (MAPK), nuclear factor-kappaB and inflammatory response in lung epithelial cells.

To investigate how respiratory epithelial cells react to an alkylating agent, we exposed human bronchial (BEAS-2B) and alveolar (A549) cells to the nitrogen mustard derivative melphalan. The BEAS-2B cells were highly sensitive to melphalan, as shown by a reduced viability after a 10-min incubation with 300 microM melphalan. The A549 cells were less sensitive and required several hours of exposure to reduce significantly in viability. However, exposure to melphalan also induces activation of intracellular signal transduction pathways, as indicated by phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38 (proteins belonging to the family of stress-induced mitogen-activated phosphorylated kinases, MAPK) within 5 min, as well as translocation of the transcription factor nuclear factor (NF)-kappaB to the nucleus within 45 min. This early activation was followed by elevated levels of tumor necrosis factor (TNF)-alpha mRNA within 2 h. We also observed increased expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of both cell lines 18 h after exposure to 25 microM melphalan and an increased adhesion of monocytes to the epithelial cells in vitro.In conclusion, we have demonstrated that alkylating compounds not only cause cell death of lung epithelial cells but also activate stress-associated MAPK signal transduction pathways and induce expression of mediators known to participate in the recruitment of inflammatory cells.

gammadelta T cells contribute to the systemic immunoglobulin E response and local B-cell reactivity in allergic eosinophilic airway inflammation.

Allergic airway inflammation induced in mice is T-cell dependent and recruitment of eosinophils to airspaces requires both alphabeta and gammadelta T cells. From previous studies it is evident that alphabeta T cells are essential for the allergic T helper type 2 (Th2)-like response, while the mechanistic contribution of gammadelta T cells is still unclear. In this study, we have investigated the role of gammadelta T cells in allergic airway eosinophilia induced by ovalbumin hypersensitivity. By comparing the responsiveness to sensitizing allergen of wild-type mice with that of T-cell receptor gammadelta knockout mice (TCRgammadelta KO) we demonstrated that mice lacking gammadelta T cells are defective in the systemic ovalbumin-specific immunoglobulin E (IgE) response. Furthermore, after aerosol challenge with allergen, gammadelta T-cell deficient mice exhibited a significantly decreased migration of B cells and natural killer cells to airways and reduced levels of allergen-specific IgG and IgA in bronchoalveolar lavage fluid. The role for B cells in the airway inflammation was indicated by the impaired ability of mice lacking functional B cells to evoke an eosinophilic response. The diminished eosinophilia in TCRgammadelta KO mice could not be explained by a defective Th2 activation since these mice displayed a normal IgG response in serum and an unaffected IG2b/IgG1 ratio in airways. Analysis of immunoregulatory cytokines in isolated lung tissue, thoracic lymph nodes and spleen further supported the notion that these mice are able to evoke a sufficient activation of T helper cells and that gammadelta T cells are not required for maintaining the Th2 profile. These results indicate that gammadelta T cells contribute to allergic airway inflammation by pathways separate from classical Th2 immune activation.