Klára Kubelková

Activation of p38 MAPK and expression of TGF-β1 in rat colon enterocytes after whole body γ-irradiation

Purpose: To examine the p38 mitogen-activated protein kinase (p38) phosphorylation and transforming growth factor beta 1 (TGF-β1) expression in rat colon enterocytes after irradiation and their contribution to pathology of intestinal radiation disease. Materials and methods: Male Wistar rats were irradiated with whole body γ-radiation of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 Gy (60Co, 1.44 Gy.min–1). Samples were taken 4 and 24 h after irradiation, immunohistochemically stained, then p38 phosphorylation and TGF-β1 expression were measured in apical and cryptal enterocytes using computer image analysis. In selected groups, morphometric parameters, mitosis and apoptosis were evaluated. Results: P38 phosphorylation integrated optical density (IOD)-based levels increased 2.4-fold (p ≤ 0.01) and 3.6 to 22.8-fold (p ≤ 0.001) in apical enterocytes 4 h after 0.5 Gy and 24 h after 3–10 Gy, respectively. TGF-β1 IOD-based expression increased 3.3- to 6.9-fold (p ≤ 0.001) and 1.6- to 4.9-fold (p ≤ 0.001) in apical cells 4 h after 0.5–2, 4, 5 Gy and 24 h after 6–10 Gy, respectively. No changes were observed in crypts. Conclusions: We found a chronological and dose-dependent order of p38 activation and TGF-β1 expression in apical enterocytes. Transient up-regulation of p38 and TGF-β1 signalling observed 4 h after low-dose irradiation may participate in molecular mechanisms creating cellular over-expression in apical compartment, while persistent patterns measured 24 h after high-dose irradiation might provide protection of remaining cells in order to maintain tissue integrity.

Specific antibodies protect gamma-irradiated mice against Francisella tularensis infection

The role of antibodies in the course of Francisella tularensis (F. tularensis) infection is still a subject of debate. The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized the model of gamma-irradiated mice for the studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses. The model of gamma-irradiated mice can also demonstrate the responses of immunocompromised host to intracellular bacterial infection. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. In this study, we demonstrate that passive transfer of immune sera protected irradiated mice against subsequent infection with strains of F. tularensis subsp. holarctica. Naïve mice of BALB/c or C3H/CBi strains were subjected to passive transfer of sera obtained from immunized mice with live vaccine strain (LVS) F. tularensis LVS, F. tularensis subsp. holarctica strain 15, heat-killed F. tularensis LVS, or heat-killed strain 15 two hours before infection with lethal doses of LVS or strain 15. The passive transfer of sera obtained from immunized mice conferred full protection of naïve unirradiated as well as sublethally irradiated mice against low lethal doses of infection with F. tularensis LVS or strain 15, in all variants of the experiments. In addition, the passively protected mice that survived the primary infection with F. tularensis LVS were protected also against further secondary challenge with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Moreover, the first evidence of combination of successful passive transfer of immunity by specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role in naïve and also in immunocompromised mice and this fact it would be appropriate to take into the account in the design of new vaccines.

Attenuation of radiation-induced gastrointestinal damage by epidermal growth factor and bone marrow transplantation in mice

Purpose: We examined the effect of epidermal growth factor (EGF) and bone marrow transplantation (BMT) on gastrointestinal damage after high-dose irradiation of mice. Material and methods: C57Black/6 mice were used. Two survival experiments were performed (12 and 13 Gy; 60Co, 0.59–0.57 Gy/min). To evaluate BMT and EGF action, five groups were established – 0 Gy, 13 Gy, 13 Gy + EGF (at 2 mg/kg, first dose 24 h after irradiation and then every 48 h), 13 Gy + BMT (5 × 106 cells from green fluorescent protein [GFP] syngenic mice, 4 h after irradiation), and 13 Gy + BMT + EGF. Survival data, blood cell counts, gastrointestine and liver parameters and GFP positive cell migration were measured. Results: BMT and EGF (three doses, at 2 mg/kg, administered 1, 3 and 5 days after irradiation) significantly increased survival (13 Gy). In blood, progressive cytopenia was observed with BMT, EGF or their combination having no improving effect early after irradiation. In gastrointestinal system, BMT, EGF and their combination attenuated radiation-induced atrophy and increased regeneration during first week after irradiation with the combination being most effective. Signs of systemic inflammatory reaction were observed 30 days after irradiation. Conclusions: Our data indicate that BMT together with EGF is a promising strategy in the treatment of high-dose whole-body irradiation damage.

Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors.

Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell–pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria’s internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

Innate Immune Recognition: Implications for the Interaction of Francisella tularensis with the Host Immune System

The intracellular bacterial pathogen Francisella tularensis causes serious infectious disease in humans and animals. Moreover, F. tularensis, a highly infectious pathogen, poses a major concern for the public as a bacterium classified under Category A of bioterrorism agents. Unfortunately, research has so far failed to develop effective vaccines, due in part to the fact that the pathogenesis of intracellular bacteria is not fully understood and in part to gaps in our understanding of innate immune recognition processes leading to the induction of adaptive immune response. Recent evidence supports the concept that immune response to external stimuli in the form of bacteria is guided by the primary interaction of the bacterium with the host cell. Based on data from different Francisella models, we present here the basic paradigms of the emerging innate immune recognition concept. According to this concept, the type of cell and its receptor(s) that initially interact with the target constitute the first signaling window; the signals produced in the course of primary interaction of the target with a reacting cell act in a paracrine manner; and the innate immune recognition process as a whole consists in a series of signaling windows modulating adaptive immune response. Finally, the host, in the strict sense, is the interacting cell.

Early cellular responses of germ-free and specific-pathogen-free mice to Francisella tularensis infection

Bacteria that are highly virulent, expressing high infectivity, and able to survive nebulization, pose great risk to the human population. One of these is Francisella tularensis, the etiological agent of tularemia. F. tularensis is a subject of intense scientific interest due to the fact that vaccines for its immunoprophylaxis in humans are not yet routinely available. One of the substantial obstacles in developing such vaccines is our insufficient knowledge of processes that initiate and regulate the expression of effective protective immunity against intracellular bacteria. Here, we present data documenting the different pattern of cellular behavior occurring in an environment unaffected by microbiota using the model of germ-free mice mono-associated with F. tularensis subsp. holarctica strain LVS in comparison with a classic specific-pathogen-free murine model during early stages of infection.

Epidermal Growth Factor Attenuates Delayed Ionizing Radiation-Induced Tissue Damage in Bone Marrow Transplanted Mice

We examined the effect of epidermal growth factor (EGF) treatment in mice that received bone marrow transplantation (BMT) after 11 Gy whole-body irradiation. C57Bl/6 mice were divided into three treatment groups: 0 Gy; 11 Gy (60Co, single dose, 0.51 Gy/min) with BMT (5 × 106 bone marrow cells isolated from green fluorescent protein syngeneic mice, 3–4 h postirradiation); and 11 Gy with BMT and EGF (2 mg/kg applied subcutaneously 1, 3 and 5 days postirradiation). Survival data were collected. Bone marrow, peripheral blood count and cytokines, gastrointestine and liver parameters and migration of green fluorescent protein-positive cells were evaluated at 63 days postirradiation. Epidermal growth factor increased survival of irradiated animals that received BMT from 10.7 to 85.7% at 180 days postirradiation. In the BMT group, we found changes in differential bone marrow and blood count, plasma cytokine levels, gastrointestinal tissues and liver at 63 days postirradiation. These alterations were completely or in some parameters at least partially restored by epidermal growth factor. These findings indicate that epidermal growth factor, administered 1, 3 and 5 days postirradiation in combination with bone marrow transplantation, significantly improves long-term prognosis.

Development of tularemic scFv antibody fragments using phage display

Polyclonal antibodies, as well as monoclonal antibodies are efficacious in providing protective immunity against Francisella tularensis. This study demonstrates the application of phage display libraries for the construction of monoclonal antibodies against F. tularensis. Novel single-chain fragment variable (scFv) antibodies were generated against a whole bacterial lysate of F. tularensis live vaccine strain using the human single fold scFv libraries I (Tomlinson I + J). A total of 20 clones reacted with the bacterial cell lysate. Further, the library contains two clones responsive to recombinant lipoprotein FTT1103Δsignal (F. tularensis subsp. tularensis Schu S4), which was constructed without a signal sequence. These positively-binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA). Then, positive scFvs were expressed in a soluble form in Escherichia coli HB2151 and tested for positive scFvs by using scFv-ELISA.