Zuzana Šinkorová

Macrophage-Assisted Inflammation and Pharmacological Regulation of the Cholinergic Anti-Inflammatory Pathway

Macrophages play an important role in the immune system. They also participate in multiple processes including angiogenesis and triggering of inflammation. The present study summarizes pieces of knowledge on the importance of macrophages in disease, especially the inflammation. Special attention is paid to the cholinergic anti-inflammatory pathway (CAP) associated with the nicotinic acetylcholine receptor (nAChR) and the parasympathetic nervous system. The current pharmacological effectiveness in suppressing the inflammation in general and the septic shock in particular, is limited. CAP was discovered recently and it seems to be a suitable target for the development of new drugs. Moreover, available drugs binding to either nAChR or acetylcholinesterase (AChE) are candidates for either an inhibition or enhancement of CAP. Though the current scientific databases do not include all necessary data on the association of CAP with body functions and the research is quite intensive, the objective of the present review is to introduce the current trends and to critically evaluate CAP and macrophage-associated pathways.

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.

Homing of lin−/CD117+ hematopoietic stem cells

In this report, we describe the homing of hematopoietic stem cells (HSCs) to non-hematopoietic tissues in lethally irradiated (9Gy) hybrid mice transplanted intravenously with lin(-)/CD117(+) bone marrow cells from ROSA26 mice. The numbers of CFU-GM in spleen of irradiated transplanted mice were well above the levels found in non-irradiated group as early as day 8 after transplant. On 12th day regeneration of lymphocytes was observed, an increase in granulocytes was detected as late as on 33rd day. Transplanted cells containing lacZ gene were detected in recipient mice by histochemistry and their location in the thymus, liver, stomach and ileum was followed during 33days post-transplantation. On 8 and 33days post-transplantation, we found massive presence of donor (lacZ(+)) cells in the thymic cortex. Hematopoietic stem cell transplantation led not only to recovery of hematopoietic and lymphoid tissues but also facilitated recovery of the small intestinal mucosa, which was significantly damaged by ionizing radiation.

Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats

Abstract Purpose: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. Materials and methods: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24–72 hours after irradiation in a dose range of 0–5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. Results: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. Conclusion: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

Soman poisoning alters p38 MAPK pathway in rat cerebellar Purkinje cells.

The aim of the study was to evaluate the expression of phosphorylated p38 mitogen‐activated protein kinase (p38 MAPK) and MAPK‐activated transcription factors elk‐1, c‐jun and c‐myc in rat cerebellar Purkinje cells after soman poisoning to investigate the pathogenetic mechanism of non‐specific long‐term adverse effects of nerve agents. Male Wistar rats were poisoned by intramuscular administration of soman at a dose 60 µg kg−1 (80% LD50), while control animals were administered physiological saline. Samples were taken 1, 7 and 14 days after poisoning, immunohistochemically stained and p‐p38MAPK, p‐c‐jun, p‐c‐myc, and p‐elk‐1 expressions were measured using computer image analysis. An increased expression of phosphorylated p38 MAPK and c‐myc 14 days after soman poisoning was found, while both activated elk‐1 and c‐jun expression remained unchanged 1, 7 and 14 days after intoxication. Late activation of p38 MAPK and their targets might be the underlying mechanism of chronic neurophysiological adverse effects. Copyright

Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

Radio-sensitization of human leukaemic molt-4 cells by DNA-dependent protein kinase inhibitor, NU7026.

In this paper we describe the influence of NU7026, a specific inhibitor of DNA-dependent protein kinase, phosphoinositide 3-kinase, and ATM-kinase on molecular and cellular mechanisms triggered by ionising irradiation in human T-lymphocyte leukaemic MOLT-4 cells. We studied the effect of this inhibitor (10 1microM) combined with gamma-radiation (1 Gy) leading to DNA damage response and induction of apoptosis. We used methods for apoptosis assessment (cell viability count and flow-cytometric analysis) and cell cycle analysis (DNA content measurement) and we detected expression and post-translational modifications (Western blotting) of proteins involved in DNA repair signalling pathways. Pre-treatment with NU7026 resulted into decreased activation of checkpoint kinase-2 (Thr68), p53 (Ser15 and Ser392), and histone H2A.X (Ser139) 2 hours after irradiation. Subsequently, combination of radiation and inhibitor led to decreased amount of cells in G2-phase arrest and into increased apoptosis after 72 hours. Our results indicate that in leukaemic cells the pre-incubation with inhibitor NU7026 followed by low doses of ionising radiation results in radio-sensitising of MOLT-4 cells via diminished DNA repair and delayed but pronounced apoptosis. This novel approach might offer new strategies in combined treatment of leukaemia diseases.

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.

Sensitivity of porcine peripheral blood leukocytes to gamma irradiation in vivo, in vitro and ex vivo.

Purpose: The large white pig is a useful experimental model to compare in vivo, in vitro and ex vivo sensitivity of peripheral blood leukocytes to ionising radiation. Such studies are impossible to perform in humans and laboratory rodents due to ethical reasons and body size, respectively. We analysed dose- and time-dependent changes of lymphocyte and granulocyte absolute numbers in porcine peripheral blood after either whole-body irradiation (in vivo and ex vivo experiments) or exposure of porcine whole blood to γ-irradiation (in vitro experiments). Materials and methods: CytoCount™ absolute counting beads and light scatter analysis using a flow cytometer were used to determine major leukocyte subpopulation numbers in blood samples after red cell removal. Results: Similar to other species, lymphocyte numbers significantly decreased in pigs both in vivo and in vitro in a dose-dependent manner. Most importantly, our data clearly show that reduction of lymphocyte numbers after irradiation in vivo proceeds much faster than after irradiation in vitro and that granulocyte changes depend only on the time of analysis after irradiation. Conclusions: All three tested experimental arrangements demonstrated the radiosensitivity of lymphocytes and the radioresistance of peripheral blood granulocytes. These in vivo and in vitro approaches, as well as the newly introduced ex vivo observations, appear to be relevant to biodosimetry.

Hematopoietic recovery after transplantation CD117+B220- (LACZ*) bone marrow cells in lethally irradiated mice.

Experiments presented here were aimed at the description of hematopoiesis repair and in vivo homing of transplanted separated CD117+B220- bone marrow cells after whole-body lethal irradiation at LD 9Gy. ROSA 26 mice were used as donors of marrow cells for transplantation [B6;129S/Gt (ROSA)26Sor] and were tagged with lacZ gene, and F2 hybrid mice [B6129SF2/J] were used as recipients of bone marrow transplanted cells. Hematopoiesis repair was provided by transplantation, both suspension of whole bone marrow cells (5x106) and isolated CD117+B220- cells (5x104). Mice survived up to thirty days after irradiation. We demonstrated that transplantation of suspension of whole bone marrow cells led to faster recovery of CFU-GM (Granulocyte-macrophage colony forming units) in bone marrow and in the spleen too. It is not clear what the share of residential and transplanted cells is in the repair process. Our results demonstrate that sufficient hematopoietic repair occurs after transplantation of CD117+B220- (lacZ+) in lethally irradiated mice, and the difference in CFU-GM numbers in the bone marrow and spleen found on day 8 posttransplant has no influence on the survival of lethally irradiated mice (30 days follow-up).