Influenza virus and factors that are associated with ICU admission, pulmonary co-infections and ICU mortality.

PURPOSE: While most influenza patients have a self-limited respiratory illness, 5-10% of hospitalized patients develop severe disease requiring ICU admission. The aim of this study was to identify influenza-specific factors associated with ICU admission and mortality. Furthermore, influenza-specific pulmonary bacterial, fungal and viral co-infections were investigated. METHODS: 199 influenza patients, admitted to two academic hospitals in the Netherlands between 01-10-2015 and 01-04-2016 were investigated of which 45/199 were admitted to the ICU. RESULTS: A history of Obstructive/Central Sleep Apnea Syndrome, myocardial infarction, dyspnea, influenza type A, BMI > 30, the development of renal failure and bacterial and fungal co-infections, were observed more frequently in patients who were admitted to the ICU, compared with patients at the normal ward. Co-infections were evident in 55.6% of ICU-admitted patients, compared with 20.1% of patients at the normal ward, mainly caused by Staphylococcus aureus, Streptococcus pneumoniae, and Aspergillus fumigatus. Non-survivors suffered from diabetes mellitus and (pre-existent) renal failure more often. CONCLUSIONS: The current study indicates that a history of OSAS/CSAS, myocardial infarction and BMI > 30 might be related to ICU admission in influenza patients. Second, ICU patients develop more pulmonary co-infections. Last, (pre-existent) renal failure and diabetes mellitus are more often observed in non-survivors.

Influences of TP53 expression on cellular radiation response and its relevance to diagnostic biodosimetry for mission environmental monitoring.

TP53 is a transcriptional activator and regulates genomic instability and cellular responses to DNA damage in response to ionising radiation. The molecular mechanism behind p53-mediated responses, such as, apoptosis and genomic instability remains unclear. An in vitro model of biological effects to irradiation was established. In order to elucidate the functional role of TP53 under different stress-reaction pathways and identify possible biological indicators, p53 was stably transfected into HL-60 cells, which provides a p53 minus background. Significantly enhanced radiosensitivity and growth suppression were observed. G(2) accumulation was obtained. Radiation-induced apoptosis of HL-60 cells was significantly inhibited by TP53, indicating that, in the event of DNA damage, TP53 is able to prevent cell death of HL-60 leukaemia cells by sustaining an arrest of the cell cycle at G(2) phase. Further evidence will be presented to identify specific radiation-targeted genes or signals as possible biomarkers for early diagnosis of radiation damage as well as mission environmental monitoring.

Irradiation differentially affects substratum-dependent survival, adhesion, and invasion of glioblastoma cell lines.

Effects of ionising radiation on extracellular matrix (ECM)-modulated cell survival and on adhesion and invasion are not well understood. In particular, the aggressiveness of glioblastoma multiforme has been associated with tumour cell invasion into adjacent normal brain tissue. To examine these effects in more depth, four human glioblastoma cell lines (A-172, U-138, LN-229 and LN-18) were irradiated on fibronectin (FN), Matrigel, BSA or polystyrene. Major findings of this study include a significantly increased survival of irradiated A-172 but not of irradiated U-138, LN-229, and LN-18 cells on FN or Matrigel compared to cells irradiated on polystyrene or BSA. Irradiation induced a dose-dependent increase in functional beta 1- and beta 3-integrins in all four glioma cell lines. This integrin induction caused improved cell adhesion to FN or Matrigel. In contrast to U-138, LN-229 and LN-18 cells, irradiation strongly impaired A-172 cell invasion. Invasion of all cell lines was inhibited by anti-integrin antibodies, the disintegrin echistatin and the MMP-2/-9 inhibitor III. Additionally, beta 1- and beta 3-integrins modulated basal and radiation-altered gelatinolytic activity of MMP-2. Tested glioblastoma cell lines showed a differential cellular susceptibility to FN or Matrigel which affected the cellular radiosensitivity. Three out of four glioma cell lines demonstrated a combination of a substratum-independent survival after irradiation and an invasive potential which was not affected by irradiation. beta 1- and beta 3-integrins were identified to play a substantial, regulatory role in survival, adhesion, invasion and MMP-2 activity. Detailed insights into radioresistance and invasion processes might offer new therapeutic strategies to enhance cell killing of lethal high-grade astrocytoma.

Comparison of the abundance of 10 radiation-induced proteins with their differential gene expression in L929 cells.

PURPOSE: To determine whether radiation-induced changes in protein abundance can be correlated with their differential gene expression in a murine fibroblast L929 cell line. MATERIALS AND METHODS: L929 cells were irradiated with 6 Gy. Cell lysates were collected at different points in time (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by two-dimensional gel electrophoresis and quantified using computerized image analysis. Proteins exhibiting a differential expression equal to or more than twofold were identified by mass spectrometry following trypsin digestion. From these, 10 proteins characterized by large changes of radiation-induced abundance were selected in order to measure their corresponding gene expression using RTQ-PCR (real-time quantitative polymerase chain reaction). RESULTS: Up to 15-fold changes in the abundance of these 10 proteins were associated with no detectable changes more than twofold on the gene expression level. However, one gene (VEGF-D) showed a significant (p=0.005) up-regulation (1.8-fold). CONCLUSIONS: Deducing protein abundance from mRNA expression levels and vice versa appears to be of limited use. Furthermore, examination of transcriptional and translational changes provides different but complementary information.

Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3beta (GSK-3beta) in vitro.

Cell adhesion to extracellular matrix (ECM) is thought to confer resistance against cell-damaging agents, that is, drugs and radiation, in tumour and normal cells in vitro. The dependence of cell survival on beta1-integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt) and glycogen synthase kinase-3beta (GSK-3beta) activity, which participate in beta1-integrin signalling and cell cycle progression was investigated as a function of radiation exposure. Colony-formation assays on polystyrene, fibronectin (FN), laminin (LA), bovine serum albumin (BSA) or poly-L-lysine (poly-L) (0-8 Gy), kinase assays, flow cytometric DNA and annexin-V analysis and immunoblotting were performed in nonirradiated and irradiated (2 or 6 Gy) A549 human lung cancer cells and CCD32 normal human lung fibroblasts. Cell contact to FN in contrast to polystyrene elevated basal ILK, PKBalpha/Akt and GSK-3beta kinase activities in A549 and CCD32 cells, as well as the basal amount of A549 G2 phase cells. Irradiation on FN or LA as compared to polystyrene, BSA or poly-L significantly improved cell survival. Following irradiation, kinase activities were stimulated strongly on polystyrene but showed to be less prominent on FN, which was because of the FN-related basal induction. Following irradiation, FN compared to polystyrene enlarged and prolonged G2 arrest in both the cell lines. For the analysis of phosphatidylinositol-3 kinase (PI3-K) dependence of protein kinases and cell cycle transition, the PI3-K inhibitors LY294002 and wortmannin were used showing decreased kinase activities, antiproliferative and radiation-dependent G2 accumulation-abrogating effects accompanied by downregulation of cyclin D1 and phospho-pRb in cells attached to polystyrene. Fibronectin partly abrogated these effects PI3-K-independently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3beta by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBalpha/Akt and GSK-3beta are involved in modulations of the cell cycle after irradiation. These interactions are strictly dependent on ECM components in a cell line-specific manner. Our findings provide molecular insights into mechanisms likely to be important for ECM-dependent cell survival and cellular radioresistance as well as tumour growth.

Proteomic analysis of radiation-induced alterations in L929 cells.

In this study we examined the protein expression profiles in X-irradiated L929 cells to get insight into how mammalian cells respond to radiation-induced cell damage. L929 cells were irradiated with the dose of 6 Gy and cell lysates were collected at different time intervals (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by 2-DE and quantified using computerized image analysis. Proteins exhibiting significant abundance alterations when comparing irradiated to unirradiated cells were identified by mass spectrometry. Using the proteomics approach we detected 47 proteins that exhibited a significant radiation-induced increase or decrease in the course of 72 h. From this group of spots 28 proteins were identified by mass spectrometry and of these 24 proteins exhibited minimally 2-fold differences in mean abundance values in comparison to controls. The identified proteins represent diverse sets of proteins participating either in protective and reparative cell responses or in induction of apoptosis and oncogenesis. The results document that proteomics is a useful method for unravelling the molecular mechanisms involved in cell reaction to ionizing radiation.

Influence of high-frequency electromagnetic fields on different modes of cell death and gene expression.

PURPOSE: International thresholds for exposure to non-ionizing radiation leading to non-thermal effects were conservatively set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). The aim of this study was to examine whether biological effects such as different modes of cell death and gene expression modifications related to tumorgenesis are detectable above the threshold defined. MATERIALS AND METHODS: Human leukaemia cells (HL-60) grown in vitro were exposed to electromagnetic fields (EMF; t 1/2(r) about 1 ns; field strength about 25 times higher than the ICNIRP reference levels for occupational exposure) leading to non-thermal effects using a high-voltage-improved GTEM cell 5302 (EMCO) connected to a pulse generator NP20 (C = 1 nF, U(Load) = 20kV). HL-60 cells were harvested at 0, 24, 48 and 72 h after radiation exposure. Micronuclei, apoptosis and abnormal cells (e.g. necrosis) were determined using morphological criteria. In parallel, the expression of 1176 genes was measured using Atlas Human 1.2. Array. Based on high data reproducibility calculated from two independent experiments (> 99%), array analysis was performed. RESULTS: No significant change in apoptosis, micronucleation, abnormal cells and differential gene expression was found. CONCLUSIONS: Exposure of HL-60 cells to EMFs 25 times higher than the ICNIRP reference levels for occupational exposure failed to induce any changes in apoptosis, micronucleation, abnormal morphologies and gene expression. Further experiments using EMFs above the conservatively defined reference level set by the ICNIRP may be desirable.

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro.

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.

Ionizing radiation modules of the expression and tyrosine phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro.

PURPOSE: Focal adhesion kinase (FAK) is involved in the regulation of many cellular processes, including cell survival and death, proliferation and migration. The same endpoints are influenced by ionizing radiation (IR). Therefore, study was performed to determine the effect of IR on the expression and phosphorylation of FAK and two of its substrates, p130cas and paxillin, in vitro. MATERIALS AND METHODS: Exponentially growing A549 lung carcinoma cells were exposed to 6 Gy X-rays. Protein expression and the extent of tyrosine phosphorylation were investigated by immunoprecipitation experiments and Western blotting analysis using specific or unspecific phosphotyrosine antibodies. Immunofluorescence staining in combination with confocal laser scanning microscopy was done to localize the proteins within the cell. RESULTS: Tyrosine phosphorylation, of Mr 110 000 150 000 and 65 000-75 000 protein bands, was induced within 30 min after exposure to IR. Three of these proteins were identified as FAK, p130cas and paxillin. IR induced phosphorylation of FAK (tyr397 and tyr925) but did not change FAK expression. Additionally, IR induced phosphorylation of paxillin (tyr31 and tyr181) within 30 min and an up-regulation of paxillin expression 2-6 h after exposure. Furthermore, a higher amount of phosphorylated p130cas was found in irradiated cells. Immunofluorescence staining demonstrated that in A549 cells, all three proteins colocalize at sites of focal adhesions at the cytoplasmic face of the cell membrane and to lamellopodia. CONCLUSIONS: The data indicate that these focal adhesion-associated proteins are modulated by IR and thus are likely to play a role in the cellular response to IR. These proteins might represent attractive targets to modulate FAK-initiated signalling pathways, which may be involved in improved radioresistance and, furthermore, in important pathological phenomena such as tumour growth and metastatic phenotypes.

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells.

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro.

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.

Fatigue, serum cytokine levels, and blood cell counts during radiotherapy of patients with breast cancer.

PURPOSE: To assess the level of fatigue during the course of adjuvant radiotherapy (RT) of breast cancer patients and its relation to anxiety, depression, serum cytokines, and blood count levels. METHODS AND MATERIALS: Forty-one patients who received adjuvant RT after breast-conserving surgery were prospectively studied. All patients underwent RT without concomitant chemotherapy. Patients rated their fatigue with two standardized self-assessment instruments, the Fatigue Assessment Questionnaire and a visual analog scale on fatigue intensity, before RT, during weeks 1-5 of RT, and 2 months after RT completion. In addition, the anxiety and depression levels were assessed with the Hospital Anxiety and Depression Scale. A differential blood cell count and the serum levels of the cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha were determined in parallel to the fatigue assessments. RESULTS: Fatigue intensity as assessed with the visual analog scale increased (p <0.001) until treatment week 4 and remained elevated until week 5. Two months after RT, the values had fallen to the pretreatment levels. Fatigue measured with the Fatigue Assessment Questionnaire did not increase significantly during treatment, but the subscores on physical (p = 0.035) and cognitive (p = 0.015) fatigue were elevated during treatment weeks 4 and 5. Affective fatigue did not change significantly. Anxiety, as rated with the Hospital Anxiety and Depression Scale, declined during RT (p = 0.002), but the Hospital Anxiety and Depression Scale depression score did not change significantly. IL-1beta, IL-6, and tumor necrosis factor-alpha levels did not change during therapy and did not correlate with fatigue. Peripheral blood cell levels declined significantly during therapy and were still low 2 months after treatment. Until treatment week 5, lymphocytes were reduced to almost 50% of their initial values. Hemoglobin levels did not correlate with fatigue. CONCLUSIONS: We observed an increase in fatigue during adjuvant RT of patients with breast cancer. Fatigue returned to pretreatment levels 2 months after treatment. No evidence was found that anxiety, depression, serum levels of IL-1beta, IL-6, tumor necrosis factor-alpha, or declining hemoglobin levels were responsible for the treatment-induced fatigue.

Proliferation kinetics and prognosis in gastric cancer after resection.

The influence of proliferation and proliferation kinetics on prognosis in gastric cancer after complete resection are controversial. In a prospective study we investigated the tumour specimens of 111 patients after resection of gastric cancer, who received 200 mg intravenous (i.v.) bromodeoxyuridine (BrdU) pre-operatively. The following biological parameters were analysed in the tumour tissue using flow-cytometry: DNA ploidy, proportion of S-phase cells, BrdU labelling index (LI), DNA synthesis time (T(s)), potential tumour doubling time (T(pot)), proliferating cell nuclear antigen (PCNA) and Ki-67 LI. The median follow-up time was 40 months (range 19-62 months). Besides the established pathohistological prognostic factors, univariate analysis revealed a prognostic influence on survival for BrdU LI, T(pot) and the proportion of S-phase cells. By multivariate Cox analysis of the completely resected cases, only tumour stage and T(pot) had a significant, independent influence on survival. By classification and regression trees (CART) analysis, resection status, tumour stage and T(pot) defined risk groups with significantly different outcomes. A short T(pot) was a predictor of better survival in stage I, II and IIIA tumours. Ploidy and the other investigated proliferation-related parameters failed to demonstrate any influence on prognosis after resection of gastric cancer.

NF-kappaB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells.

PURPOSE: To investigate the mechanisms leading to initiation by ionizing radiation of IL-6 transcription in HeLa cells. MATERIALS AND METHODS: HeLa cells were irradiated with X-rays at a dose rate of approximately 1 Gy/min or treated with TPA (100 ng/ml). Transient transfection analysis with truncated IL-6 promoter CAT constructs was used to identify the radiation-sensitive region within the IL-6 promoter/enhancer. RESULTS: For basal expression of the IL-6 gene in unirradiated control cells the presence of the binding site for the nuclear factor kappa B (NF-kappaB) and the multiple response elements (MRE) were necessary. After deletion of either the activator protein (AP)-1 or the MRE site, radiation-induced IL-6 promoter CAT activity was significantly reduced, whereas after deletion of the NF-kappaB site it was completely abolished. Maximal radiation-induced IL-6 promoter CAT activity was observed when the AP-1, NF-kappaB and MRE motifs were present. In electrophoretic mobility shift analyses (EMSA), X-ray-inducible activity was found for NF-kappaB and AP-1 at the MRE constitutive, but no inducible activities were detectable. The nuclear factor IL-6 (NF-IL6) element showed no specific radiation-responsive activity. CONCLUSIONS: These results demonstrate that NF-kappaB plays a major role in X-ray-inducible IL-6 expression in HeLa cells. The fact that IL-6 promoter activity was dramatically enhanced in the presence of the MRE and distal AP-1 binding motif is indicative of a cooperative mode of transcriptional activation involving all three transcription factor systems. These data provide new insights into the prodromal events of radiation-induced inflammation of epithelial cells and putatively the cutaneous radiation syndrome.

Correlation of micronucleus and apoptosis assays with reproductive cell death can be improved by considering other modes of death.

PURPOSE: To evaluate, using 10 cell lines, the already shown improved correlation between the sum of micronucleated plus apoptotic cells and reproductive cell death. MATERIALS AND METHODS: The cell lines were X-irradiated with doses selected to obtain comparable survival levels. Micronucleated and apoptotic cells as well as abnormal cells (multinucleated and necrotic cells) were counted over several days. Apoptosis was also confirmed by gel electrophoresis. RESULTS: The data confirmed the already shown improved relationship over the solely performed micronucleus or apoptosis assays with reproductive cell death and radiation dose. However, even this approach revealed a saturation after irradiation in certain cell lines. Including other modes of cell death such as mitotic failure or necrosis could eliminate this effect. CONCLUSION: Multiple parameters should be considered when evaluating the use of a predictive assay for ionizing radiation-induced cell killing or biological dosimetry.

Estimating medical resources required following a nuclear event.

Large quantities of medical resources are necessary for the treatment of patients suffering from acute radiation syndrome in combination with blast and thermal injuries (combined injuries). So far, however, there is no tool available for evaluating the resources required for the treatment of combined injuries. The purpose of this manuscript is to describe the scientific basis for a newly developed software tool designed to estimate the medical resources needed for treating the combined injuries of individuals/high numbers of casualties who may be involved in civil defense, emergency medical care and various military activities (namely out of area) in the case of a nuclear event.

Apoptosis in human embryonal cell carcinoma: preliminary results.

Disorders in the regulation of apoptotic cell death may contribute to cancer. Furthermore, lymphocytes are supposed to play a role in counteracting tumorigenesis by inducing apoptosis in different human tumors. In this study, for the first time, tumor cell and lymphocyte apoptosis were investigated systematically in human embryonal cell carcinoma. DNA fragmentation and DNA condensation were measured simultaneously on double-fluorescence-labeled testis tumor sections using immunofluorescence microscopy. Different apoptotic indices (AIs), based either on biochemical (DNA fragmentation) or morphological criteria (DNA condensation) alone or on a combination of both, were determined in different histological regions in and around the tumor. Using morphological criteria alone, 40-75% of all apoptotic cells were not detected. Based on previous observations this finding might be related to subsets of apoptotic cells which induce the process of DNA condensation without activation of processes responsible for DNA fragmentation. Moreover, the AIs of tumor cells and lymphocytes were highest in the tumor region, compared with regions around the tumor and distant from it; these findings are discussed in the context of the Fas/FasL system.

Influence of clinically used antioxidants on radiation-induced expression of intercellular cell adhesion molecule-1 on HUVEC.

PURPOSE: The effects of antioxidants (N-acetyl-L-cysteine [NAC] and pyrrolidine dithiocarbamate [PDTC]) on radiation-induced ICAM-1 expression on human umbilical vein endothelial cells (HUVEC) were investigated. MATERIALS AND METHODS: The expression of ICAM-1 on HUVEC was determined by flow cytometry up to 72 h after X-irradiation. Functional competence of induced ICAM-1 was assessed by adhesion experiments with human polymorphonuclear neutrophils on irradiated HUVEC. RESULTS: Preincubation of cells with both or either NAC and PDTC was unable to reduce radiation-induced ICAM-1 expression on HUVEC. In fact, by themselves, these antioxidants induced a significant increase of ICAM-1 expression, which in comparison with a radiation dose of 7 Gy after 24h was nine times higher for PDTC, and more than double for NAC. Treatment with NAC clearly restrained TNF-alpha-induced ICAM expression on HUVEC, while preincubation of cells with PDTC showed synergistic effects. CONCLUSIONS: The role of reactive oxygen intermediates in signal transduction pathways leading to ICAM-1 expression should be investigated further. Furthermore, antioxidants may exert a pro-inflammatory role, as revealed by the induction of ICAM-1 expression on endothelial cells. The inhibition of TNF-alpha-induced ICAM-1 expression by NAC might have clinical implications because this substance is used as a radioprotector in radiotherapy.

Modulation of signal transduction pathways and global protein composition of macrophages by ionizing radiation.

It is assumed that the exposure of cells to ionizing radiation modulates their signal transduction pathways, which then govern the early and late radiation-induced alterations in gene expression. In this study we tested the effects of low doses of X-irradiation on the cell signaling and global protein composition of an HL-60 human promyelocytic leukemia cell line differentiated along a macrophage-like cell pathway by 4beta-phorbol-12-myristate-13-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunoprecipitates, we found radiation-induced changes in the level of phosphorylation of proteins with molecular masses of 45 and 48 kDa, but in the most intensively stained area, ranging from 54 to 60 kDa, no alterations were observed. When two-dimensional electrophoresis (2-DE) immunoblotting was applied, only proteins from this heavily stained region were visualized and in addition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Furthermore, the immunostaining of extracellular signal-regulated kinase 2 (ERK2) which did not prove its tyrosine phosphorylation demonstrated the existence of several ERK2 charge isoforms showing differential expression after X-irradiation. Comparing the whole protein profiles we found after the simultaneous quantitation of 1000 matched spots two proteins whose expression was regulated in an opposite manner in nonirradiated and X-irradiated cells. The quantities of both spots showed increases or decreases by a factor of 2 or more between irradiated and nonirradiated samples and both these changes were statistically significant (P<0.05).

Tissue engineering with HaCaT cells and a fibroblast cell line.

Most skin models consist of primary cells. Our aim was to develop a highly reproducible skin model consisting only of cell lines to investigate irradiation effects. The spontaneously immortalized human keratinocyte line HaCaT is known for its capacity for epidermal differentiation. As an organotypic coculture, HaCaT cells were grown air-exposed on top of a dermis equivalent consisting of a murine fibroblast cell line (L929) in collagen. The technique for the preparation of this coculture system is described. After 3 weeks a multilayered epithelium with signs of differentiation developed. The expression of several markers for differentiation and basal membrane formation were compared with those of healthy human skin by immunohistochemical staining. In the epithelium of the skin model several cytokeratins, especially keratin 10, and involucrin were expressed comparable to normal skin. Laminin expression was found along the basal zone of the epithelium. BrdU labeling indicating proliferation was mainly found in the basal parts of the epithelium. Differentiated cells showing DNA fragmentation were detected in the upper parts of the epithelium by the TUNEL assay. Fluorescence in situ hybridization was used to discriminate between HaCaT and L929 cells in the coculture. Some L929 cells growing on top of the epithelium could be detected. This might have been due to an invasion of highly proliferating L929 cells and might be one of the limits of tissue engineering with cell lines. In conclusion, the organotypic coculture used as a skin model is a promising additional tool for addressing specific research questions.