Susceptibility of porcine peripheral blood to ionizing radiation in vivo and in vitro experiments.

Absolute counts of porcine peripheral blood cells were quantified by flow cytometry as a marker of radiosensitivity. Irradiated human organism responds to irradiation among others by declining haemopoiesis and inducing an apoptosis of radiosensitive cells, especially lymphocytes. However can be changes of counts of human peripheral blood cells well examinated within a model in vitro, in vivo model is not obtainable in adequate range of doses. The experimental model of one month old large white pig was used to compare effects in these two systems. Young piglets were whole-body-irradiated and assessed in dependence on time upon irradiation. For the dose-response analyses doses 0-2-4-6 and 10 Gy were applied at 0,4 Gy/min (60Co gamma-rays). Beside that heparin-treated peripheral blood was irradiated apart and analysed at the same time frequency. Absolute counts of leukocyte populations were analysed by CytoCountTM technique (DakoCytomation) applying a suspension of fluorescent microspheres as a reference population for enumeration of lymphocytes, granulocytes and monocytes which were identificated using a flow cytometry through gating within forward scatter versus side scatter. Counts of cells were expressed as a ratio of individual population with regard to nonirradiated negative control sample. The estimated decrease of lymphocytes manifested more intensive within in vivo experoments than in vitro. The maximum downtrend of both was marked during eight hours after irradiation. Decrease of granulocytes was very low with no distinct response to irradiation within system in vitro whereas analyses in vivo documented intensive release of granulocytes from body reserves into peripheral blood as a typical inflammation reaction. Populations of monocytes were hardly detectable and were not included in our study. Nevertheless variance in absolute counts found out in system in vivo versus in vitro after irradiation showed irretrievability of using in vivo system within radiosensitivity studies. This work was supported by grant of Ministry of Defence No. OPUOFVZ200604 and by grant of Ministry of Education, Youth and Sports No.2B08028

Pig as an experimental model for in vitro and in vivo studies of radiosensitivity of B lymphocytes and NK cell

Introduction: Lymphocyte subsets differ in their susceptibility to ionizing radiation. B cells are more radiosensitive than major (CD4+CD8- and CD4-CD8+) T cell subsets; results on radiation-induced NK cell apoptosis are controversial. Most data come either from experiments in vitro or examinations upon accidental irradiation with ill-defined doses. In vivo models include laboratory rodents and a large animal model is missing. Here we show that pigs represent a convenient model for in vitro and vivo studies of radiosensitivity of B lymphocytes subsets with possible implications in biodosimetry. Methods: Doses in the range of 1-8 Gy were used for whole body irradiation of one-month-old boars. Heparinized blood was collected before irradiation (control), immediately after irradiation and after selected time intervals (up to 48 hr). Samples were analyzed immediately after collection or upon 6 - 48 hr cultivation. Total leukocyte counts and relative numbers of lymphocyte subsets were analyzed by immunophenotyping and multicolor flow cytometry. Results: As expected, total leukocytes counts decreased with a dose and time both in vivo and in vitro. Similar to humans and rodents, B cells represented the most sensitive lymphocyte population. The comparison of individual B cell subsets has revealed that the CD2- subset is less prone to radiation induced apoptosis than CD2+ B cells. In the T cell compartment, CD4+CD8- cells are more radioresistant than their CD4-CD8+ counterparts. Among CD4+ T cell subsets, the double positive (CD4+CD8+) population and cells with surface CD25 expression belong to the most radioresistant ones. Importantly, while the CD8+ NK population (CD3-CD8+ lymphocytes) rapidly disappears after irradiation both in vitro and in vivo, such cells re-appeared circulation as soon as 24 hr after irradiation with lower doses. Conclusions: Short term (6-24 hr) cultivation of full blood collected from irradiated individuals represents a convenient and reliable approach for in vivo studies on ionizing radiation induced apoptosis of lymphocyte subsets in vivo. B and NK subsets have been proved as useful biomarkers of the received dose. Different radiosensitivity of NK cells and their precursors is suggested as the reason of formerly published discrepancies in NK cells radiosensitivity. Acknowledgement: Supported by projects of the Ministry of Defence of the CR OPUOFVZ200604 and Ministry of Education,Youth and Sports No. 2B08028

Molecular mechanisms of response of human peripheral blood mononuclear cells to ionizing radiation

Introduction: The aim of this study was to compare reaction of quiescent and proliferating PHA (mitogenic lectin phytohemagglutinin)-stimulated human peripheral blood mononuclear cells (PBMCs) to γ-radiation (IR) and analyze changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53 and its phosphorylations on serine 15 and 392, and p21. Materials and methods: PBMC were acquired from venous blood of normal donors by centrifugation over Histopague. The lymphocytes were cultured in IMDM medium suplemented with 20% fetal calf serum and antibiotics. For mitogenic stimulation of lymphocytes was added 10 μg/ml PHA. The viability of the PBMC has been identified by flow cytometry. Western blotting has been used for p53 and its phosphorylation form, p21 and H2AX detection. T-lymphocytes and PBMC were irradiated and lysed. To enhance detection sensitivity the Total p53 and PathScan p53ser15 Sandwich ELISA Kits were used. Results: PBMCs were stimulated to enter the cell cycle by treatment with the PHA. After 72 h long stimulation by PHA 96 % of CD3+cells were CD25+ and 12 % entered cell cycle (S+ and G2 phase). PHA-stimulation itself causes increase in γH2A.X, p53, and p21, but not phosphorylation of p53. After the irradiation of these stimulated PBMCs we detected increase in p53 and its phosphorylations on serine 15 and 392, and further increase in p21 from 4 h after the irradiation. Also level of γH2A.X increased significantly. Increase of p53 phosphorylation on serine 15 is dose-dependent 4 h after the irradiation in the whole studied dose range (0.5-7.5 Gy) in stimulated PBMCs. We compared p53, p53ser15 and ser392, p21 and H2AX between grouply of phytohemaglutinin-stimulated and non-stimulated PBMC 4 hours after 0,5-7,5Gy as well as 1-72 hours after 4Gy irradiation.. We were unable to detect p53 accumulation or phosphorylation in response unstimulated PBMC to gamma radiation induced DNA damage. We induced cell cycling by phytohemaglutinin in the T-cell fraction of PMBC preparations. Althougly total p53 protein expression was unchanged after 4 Gy irradiation, we found p53ser392 time-dependent expression (1-72 hours, 4 Gy) and p53ser15 dose-dependent expression in range of 0,5-7,5 Gy 4 hours after irradiation in phytohemaglutinin- stimulated PBMC. Neither total p53 protein nor p53ser392 was not altered by irradiation in non-stimulated PBMC. Conclusions: We suggested that p53 protein accumulation is a common mechanism for induction apoptosis of irradiated cells. We suggest that this pathway is activated in proliferating lymphocytes, unlike quiescent lymphocytes

Caffeine inhibits ATM-dependent phosphorylation of p53 in gamma-irradiated leukaemic MOLT-4 cells.

Objective: Activation of ATM-kinase (ATM) is involved in cell cycle arrest and reparation of ionizing radiation (IR)-induced DNA damage. Caffeine, a non-specific inhibitor of ATM, shortens IR-induced G2 arrest. It was shown to inhibit ATM/Chk-2/p53 signalling pathway and to diminish DNA reparation of IR-induced double strand breaks. In this work we studied effect of ATM inhibition on activation of other proteins involved in induction of apoptosis and cell cycle control in human T-lymphocyte leukaemic MOLT-4 cells (p53 wild type). We evaluated expression of tumour-suppressor p53 (itself and phosphorylated at Ser-15 and Ser-392) and p21 (a cell cycle regulator). Methods: We added 2mM caffeine 30 min prior irradiation (60Co) and washed out after 24 h. The cells were irradiated by the doses of 1 or 3 Gy, lysed and the proteins were separated by SDS-PAGE and detected by Western-blotting. Apoptosis (annexin V detection) and DNA content were determined by flow-cytometry. Results: Protein p53 was up-regulated 2 h after irradiation by the doses of 1 and 3 Gy with maximum after 16 h. After 24 h its amount decreased. Both doses of IR induced phosphorylation at both Ser-15 and Ser-392 after 2 h with maximum after 4 h. Adding caffeine significantly inhibited both p53 phosphorylations. Similarly, p21 was up-regulated 4 h after irradiation by the doses of 1 and 3 Gy with maximum after 24 h. Caffeine caused a substantial decrease of p21 in combination with both doses of IR. Three days after irradiation caffeine significantly potentiated induction of apoptosis. Conclusion: Caffeine significantly inhibited ATM/Chk-2/p53 signalling pathway in MOLT-4 cells, which via decreased expression of p21 led to inhibition of cyclin-dependent kinases. This resulted in shortened cell cycle arrest (necessary for effective DNA reparation) and induction of apoptosis. Therefore we conclude that ATM activity inhibitors such as caffeine have a radio-sensitising effect and could be exploited in radio-therapy as radio-sensibilisators. Acknowledgement: The authors acknowledge the financial support of Ministry of Education of Czech Republic (project no. MSM 0021620820) and Ministry of Defence of Czech Republic (project no. MO0FVZ0000501)

Gut microbiota-host interactions and juvenile idiopathic arthritis

Background: Juvenile idiopathic arthritis is the most common form of chronic arthritis in children. There is mounting evidence that the microbiota may influence the disease. Main body: Recent observations in several systemic inflammatory diseases including JIA have indicated that abnormalities in the contents of the microbiota may be factors in disease pathogenesis, while other studies in turn have shown that environmental factors impacting the composition of the microbiota, such as delivery mode and early exposure to antibiotics, affect the risk of chronic inflammatory diseases including JIA. Microbial alterations may predispose to JIA through a variety of mechanisms, including impaired immunologic development, alterations in the balances of pro- versus anti-inflammatory bacteria, and low-grade mucosal inflammation. Additional confirmatory studies of microbiota aberrations and their risk factors are needed, as well as additional mechanistic studies linking these alterations to the disease itself. Conclusions: The microbiota may influence the risk of JIA and other systemic inflammatory conditions through a variety of mechanisms. Additional research is required to improve our understanding of the links between the microbiota and arthritis, and the treatment implications thereof