Michal Kroca

The immunomodulatory effect(s) of lead and cadmium on the cells of immune system in vitro

A number of studies documented that the heavy metals are not only toxic for the organisms but they may modulate immune responses. The immunomodulatory activity was proved in several in vivo and in vitro model systems. In the current study, immunomodulatory activities of lead and cadmium are presented. The viability of both lymphocytes and macrophages was affected by heavy metals in a dose- and time-dependent manner. In the case of lead, the depression of N-oxide production closely correlated with increased blast transformation of spleen cells induced by concanavalin A (ConA). On the contrary, cadmium suppressed the production of N-oxides but stimulated significantly the proliferation of spleen cells. The production of cytokines by lymphocytes and macrophages was dependent on the in vitro model used. Generally, the treatment of macrophages with lead results in disregulation of the production of proinflammatory cytokines [tumour necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha) and interleukin 6 (IL-6)] and preferential production of Th1 type of cytokines (IFN-gamma and IL-2). Cadmium seemed to trigger the Th2 cytokine regulatory pathway [interleukin 4 (IL-4), interleukin 10 (IL-10)]. The results suggest the metal-induced changes in immunoregulatory mechanism of host with potentially severe clinical consequences.

Proteome Analysis of an Attenuated Francisella tularensis dsbA Mutant: Identification of Potential DsbA Substrate Proteins

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two‐dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two‐dimensional gel electrophoresis with immobilized pH 3–10 gradient in the first dimension and 9–16% gradient or tricine SDS‐PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Interaction of B cells with intracellular pathogen Francisella tularensis

Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.

Production of stress-inducible form of heat-shock protein 70 in mouse peritoneal adherent cells after in vivo infection by Francisella tularensis.

Heat-shock proteins (hsp) are ubiquitously produced molecules which participate in the protection of cells from environmental perturbation. Moreover, the members of the heat-shock protein 60 (hsp60) and 70 (hsp70) families play an important role in pathogen-host interactions. We studiedin vivo production of the 70-kDa heat-shock proteins in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with an attenuated vaccine strain (LVS) ofFrancisella tularensis. We found a differential production of a highly stress-inducible member of the hsp70 family, designated hsp72, in three inbred strains of mice exhibiting either resistance or susceptibility toF. tularensis LVS infection. Whereas in tularemia-resistant mice hsp72 was even expressed in PEC without injection of bacteria and its production further increased on day 3 and slowly declined on days 5 and 7 after injection, in susceptible mice hsp72 production was highly inducble and restricted only to day 3 afterin vivo infection. Further analysis of hsp72 expression revealed intracellular hsp72 accumulation and its preferential production by peritoneal adherent cells.

Measurement of Biological Aerosols in an Aerosol Chamber

A standard system for the aerosol exposure of mice with viable Francisella tularensis live vaccine strain (LVS) in an aerosol chamber was developed. The pneumatic nebulizer and the dynamic “nose only” aerosol chamber for exposure of 12 mice were used. Pressure of the orifice and flow rate through the atomizer, flow rate of the air, temperature, and humidity in the chamber were measured. Density, viscosity, consumption of the aerosolized suspension, and the number of cultivable bacteria in the suspension were measured. The correlation between the technical parameters and the number of the cultivable bacteria in the initial suspension, aerosol, and the murine lungs were determined.