The influence of low-level sarin inhalation exposure on the host resistance and immune reaction of inbred BALB/c mice after their infection with Francisella tularensis LVS.

To study the influence of low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to two low concentrations of sarin for 60 minutes in the inhalation chamber and then infected with Francisella tularensis LVS on the 7th day following the exposure to sarin. 24 hours after infection, the level of some isotypes of antibodies (IgM, IgA) against tularaemia was significantly decreased regardless of the sarin concentration used while the lymphoproliferation was significantly increased regardless of the mitogen and sarin concentration used. Later, the level of some isotypes of antibodies (IgM, IgA) against tularaemia and the vitality of Francisella tularensis LVS was significantly increased in the case of exposure of mice to clinically symptomatic concentration of sarin (7 days after infection) while the lymphoproliferation was significantly decreased regardless of the concentration of sarin when specific tularaemic antigen Ag4 was used as a mitogen (3 weeks after infection). Thus, the results indicate that not only symptomatic but also asymptomatic dose of sarin is able to alter the host resistance and reaction of immune system, especially at 24 hours and 7 days following infection with Francisella tularensis LVS. Nevertheless, the alteration of immune functions following the inhalation exposure to a symptomatic concentration of sarin seems to be more pronounced.

The influence of single or repeated low-level sarin exposure on immune functions of inbred BALB/c mice.

To study the influence of single or repeated low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low clinically asymptomatic concentrations of sarin for 60 min. in the inhalation chamber. The evaluation of immune functions was carried out using phenotyping of CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD19 (B-lymphocytes) in the lungs, blood and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages and the measurement of the natural killer cell activity at one week after sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that an asymptomatic dose of sarin is able to alter the reaction of the immune system at one week after exposure to sarin. While the number of CD3 cells in lung was significantly decreased, a slight increase in CD19 cells was observed especially in the lungs after a single sarin inhalation exposure. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used and the number of low-level sarin exposures. The ability of peritoneal and alveolar macrophages to phagocyte the microbes was also decreased regardless of the number of low-level sarin exposures. The production of N-oxides by peritoneal macrophages was decreased following a single low-level sarin exposure but increased following repeated low-level sarin inhalation exposure. Nevertheless, the changes in the production of N-oxides that reflects a bactericidal activity of peritoneal macrophages was not significant. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to low concentration of sarin regardless of the number of exposures. Thus, not only organophosphorous insecticides but also nerve agents such as sarin are able to alter immune functions following a single inhalation exposure even at a dose that does not cause clinically manifested intoxication. Generally, the repeated exposure to low concentrations of sarin does not increase the alteration of immune functions compared to the single low-level sarin exposure with the exception of phagocyte activity of alveolar macrophages and natural killer cell activity.

Low-level sarin-induced alteration of immune system reaction in inbred BALB/c mice.

To study the influence of low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low concentrations of sarin for 60 min in the inhalation chamber. Two concentrations of sarin were chosen-asymptomatic concentration (LEVEL 1) and non-convulsive symptomatic concentration (LEVEL 2). The evaluation of immune functions was carried out using phenotyping of CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD19 cells (B-lymphocytes) in the lungs, blood and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages and the measurement of the natural killer cell activity at 1 week following sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that not only symptomatic but also asymptomatic dose of sarin is able to alter the reaction of immune system at 1 week following exposure to sarin. While the number of CD3 cells in the lungs was slightly decreased, an increase in CD19 cells was observed especially in the lungs and blood. The reduced proportion of T-lymphocytes is caused by decay of CD4 positive T-cells. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used. The production of N-oxides by peritoneal macrophages was stimulated after exposure to LEVEL 2 of sarin whereas their ability to phagocyte the microbes was increased after exposure to LEVEL 1. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to LEVEL 2 of sarin. Thus, not only organophosphorus insecticides but also nerve agents such as sarin are able to alter immune functions even at a dose that does not cause clinically manifested intoxication following the inhalation exposure. Nevertheless, the alteration of immune functions following the inhalation exposure to a symptomatic concentration of sarin seems to be more pronounced.

Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis.

The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8(+) T-cell epitope.

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.