Cytokines and radiation-induced pulmonary injuries.

Radiation therapy is one of the most common treatment strategies for thorax malignancies. One of the considerable limitations of this therapy is its toxicity to normal tissue. The lung is the major dose-limiting organ for radiotherapy. That is because ionizing radiation produces reactive oxygen species that induce lesions, and not only is tumor tissue damaged, but overwhelming inflammatory lung damage can occur in the alveolar epithelium and capillary endothelium. This damage may result in radiation-induced pneumonitis and/or fibrosis. While describing the lung response to irradiation generally, the main focus of this review is on cytokines and their roles and functions within the individual stages. We discuss the relationship between radiation and cytokines and their direct and indirect effects on the formation and development of radiation injuries. Although this topic has been intensively studied and discussed for years, we still do not completely understand the roles of cytokines. Experimental data on cytokine involvement are fragmented across a large number of experimental studies; hence, the need for this review of the current knowledge. Cytokines are considered not only as molecular factors involved in the signaling network in pathological processes, but also for their diagnostic potential. A concentrated effort has been made to identify the significant immune system proteins showing positive correlation between serum levels and tissue damages. Elucidating the correlations between the extent and nature of radiation-induced pulmonary injuries and the levels of one or more key cytokines that initiate and control those damages may improve the efficacy of radiotherapy in cancer treatment and ultimately the well-being of patients.

New Light on An Old Friend: Targeting PUMA in Radioprotection and Therapy of Cardiovascular and Neurodegenerative Diseases.

This review summarizes recent progress in understanding the role of p53-upregulated mediator of apoptosis (PUMA) in molecular pathways with respect to its potential therapeutic applications. Particular emphasis is given to the PUMA´s role in ionizing radiation-induced signalling as radiotoxicity of normal tissue is mediated mostly via apoptosis. PUMA and its p53-dependent and p53- independent induction are described and potential use as a new target for the development of radioprotective agents is suggested. Further implications, including targeting PUMA to prevent and treat cardiovascular and neurodegenerative diseases, are also discussed together with an overview of other therapeutic applications. Finally, basic chemical structures for the development of novel PUMA modulators such as pifithrine derivatives, kinase inhibitors or modulators of Bcl-2 protein family are described.

The first in vivo multiparametric comparison of different radiation exposure biomarkers in human blood.

The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.

Cold Plasma and Acid Treatment Modification Effects on Phonolite.

A sample of phonolite was treated by cold plasma and hydrochloric acid diluted in water to study the change of its structure and acid properties. The phonolite and treated samples were analysed by XRD, elemental analysis XRF, specific surface area BET, TPD-NH3 and FT-IR spectroscopy. They were also tested in the adsorption of Ca, K, Mg, P and Na impurities present in waste cooking oil. Plasma treated sample presented almost the same structure with some surface differences respect to the original phonolite. However, acid treated sample presented bigger total surface compared to the other samples, different structure, composition and acid properties.

B cell subsets are activated and produce cytokines during early phases of Francisella tularensis LVS infection.

Francisella tularensis, a facultative intracellular Gram-negative bacterium, causes the illness tularemia. The infection of mice with live vaccine strain is considered to be a model of human tularemia. F. tularensis infects predominantly such phagocytic cells as macrophages or neutrophils, but it also infects non-phagocytic hepatocytes, epithelial cells, and murine and human B cell lines. Based on work with the murine tularemia model, we report here that F. tularensis LVS infects peritoneal CD19(+) cells - exclusively B-1a cells - early after intraperitoneal infection in vivo. The peritoneal and consequently spleen CD19(+) cells are activated by the F. tularensis LVS infection to express the activation markers from MHC class II, CD25, CD54, CD69, and the co-stimulatory molecules CD80 and CD86. As early as 12 h post-infection, the peritoneal CD19(+) cells produce IFN-γ, IL-1ß, IL-4, IL-6, IL-12, IL-17, IL-23, and TNF-α. The spleen CD19(+) cells respond to infection with some delay. Moreover, the F. tularensis infected A20 B cell line activates CD3(+) spleen cells isolated from naïve mice. Thus, the data presented here suggest that B cells have all the attributes to actively participate in the induction and regulation of the adaptive immune response during early stages of F. tularensis infection.

Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor, NU7441.

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 µM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60).

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 µM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 µM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 µM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 µM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

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

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.

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.

Activation of p38 MAPK and expression of TGF-ß1 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-ß1) 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 ((60)Co, 1.44 Gy.min(-1)). Samples were taken 4 and 24 h after irradiation, immunohistochemically stained, then p38 phosphorylation and TGF-ß1 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-ß1 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-ß1 expression in apical enterocytes. Transient up-regulation of p38 and TGF-ß1 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.

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.

Exposure to fractionated dose of 60 Gy affects molecular response of HL-60 cells to irradiation.

In this work we evaluated changes in molecular response of human promyelocyte leukemia cells HL-60 and HL-60-IR cells (HL-60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months) to irradiation by the dose of 2 and 8 Gy. Analysis of CD11b and apoptosis by flow-cytometry revealed that on 3rd day after irradiation by 8 Gy the HL-60-IR are more resistant to radiation-induced apoptosis and more differentiated (increase in CD11b in non-apoptotic cells) than regular HL-60. We found that both types of cells have high basal level of phosphorylated extracellular signal-regulated kinases Erk1/2 . Irradiation induces decrease in Erk1/2 phosphorylation after 4 and 8 h in both cell types. However, in HL-60-IR cells Erk1/2 phosphorylation is restored faster than in HL-60. Also it was found that in contrary to HL-60 cells, the HL-60-IR cells react to 2 Gy irradiation by p53 independent increase in p21(WAF1/Cip1), and not by activation of checkpoint kinase Chk-2. Therefore we concluded that relatively high dose of radiation (6 Gy) does not lead after 10 repetitive irradiations to eradication of HL-60 cells, but instead increases their radioresistance, increases the ability to differentiate, alters MAPK response, increases amount of p21(WAF1/Cip1), and decreases induction of apoptosis by ionizing radiation. p21(WAF1/Cip1) might prevent apoptosis induction and trigger permanent cell-cycle arrest, which can also contribute to regression of this leukaemia after therapy.

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 microg kg(-1) (80% LD(50)), 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.