A COMPARISON BETWEEN X-RAY AND CARBON ION IRRADIATION IN HUMAN NEURAL STEM CELLS.

Glioblastoma multiforme (GBM) is characterized by a poor prognosis and a median survival of ~12-18 months. GBM is usually managed by neurosurgery followed by both chemotherapy and radiotherapy. Since GBM develops resistance to conventional therapies, treatment with C-ions is promising to completely eradicate the tumoural mass. During cranial irradiation, exposure of healthy tissues is inevitable. Because of the presence of neural stem cells, a deep investigation on the effects of C-ion irradiation with respect to X-ray induced damage is mandatory to allow a better definition of treatments. In this work, the comparison of X-rays and C-ion irradiation-induced effects on human neural stem cell, focusing on multiple endpoints, such as cell viability, cytokine secretion and spheroid formation is presented. Results show different temporal and dose responses of human neural stem cells to the different radiation qualities, suggesting different underpinning mechanisms of radiation-induced damages.

WHAT ROLES FOR TRACK-STRUCTURE AND MICRODOSIMETRY IN THE ERA OF -omics AND SYSTEMS BIOLOGY?

Ionizing radiation is a peculiar perturbation when it comes to damage to biological systems: it proceeds through discrete energy depositions, over a short temporal scale and a spatial scale critical for subcellular targets as DNA, whose damage complexity determines the outcome of the exposure. This lies at the basis of the success of track structure (and nanodosimetry) and microdosimetry in radiation biology. However, such reductionist approaches cannot account for the complex network of interactions regulating the overall response of the system to radiation, particularly when effects are manifest at the supracellular level and involve long times. Systems radiation biology is increasingly gaining ground, but the gap between reductionist and holistic approaches is becoming larger. This paper presents considerations on what roles track structure and microdosimetry can have in the attempt to fill this gap, and on how they can be further exploited to interpret radiobiological data and inform systemic approaches.

AT THE PHYSICS-BIOLOGY INTERFACE: THE NEUTRON AFFAIR.

We present predictions of neutron relative biological effectiveness (RBE) for cell irradiations with neutron beams at PTB-Braunschweig. A neutron RBE model is adopted to evaluate initial DNA damage induction given the neutron-induced charged particle field. RBE values are predicted for cell exposures to quasi-monoenergetic beams (0.56 MeV, 1.2 MeV) and to a broad energy distribution neutron field with dose-averaged energy of 5.75 MeV. Results are compared to what obtained with our RBE predictions for neutrons at similar energies, when a 30-cm sphere is irradiated in an isotropic neutron field. RBE values for experimental conditions are higher for the lowest neutron energies, because, as expected, target geometry determines the weight of the low-effectiveness photon component of the neutron dose. These results highlight the importance of characterizing neutron fields in terms of physical interactions, to fully understand neutron-induced biological effects, contributing to risk estimation and to the improvement of radiation protection standards.

The origin of neutron biological effectiveness as a function of energy.

The understanding of the impact of radiation quality in early and late responses of biological targets to ionizing radiation exposure necessarily grounds on the results of mechanistic studies starting from physical interactions. This is particularly true when, already at the physical stage, the radiation field is mixed, as it is the case for neutron exposure. Neutron Relative Biological Effectiveness (RBE) is energy dependent, maximal for energies ~1 MeV, varying significantly among different experiments. The aim of this work is to shed light on neutron biological effectiveness as a function of field characteristics, with a comprehensive modeling approach: this brings together transport calculations of neutrons through matter (with the code PHITS) and the predictive power of the biophysical track structure code PARTRAC in terms of DNA damage evaluation. Two different energy dependent neutron RBE models are proposed: the first is phenomenological and based only on the characterization of linear energy transfer on a microscopic scale; the second is purely ab-initio and based on the induction of complex DNA damage. Results for the two models are compared and found in good qualitative agreement with current standards for radiation protection factors, which are agreed upon on the basis of RBE data.

Mechanisms of the induction of apoptosis mediated by radiation-induced cytokine release.

The aim of the present work was to investigate the mechanisms of radiation-induced bystander signalling leading to apoptosis in non-irradiated co-cultured cells. Cultured non-transformed cells were irradiated, and the effect on the apoptosis rate on co-cultured non-irradiated malignant cells was determined. For this, two different levels of the investigation are presented, i.e. release of signalling proteins and transcriptomic profiling of the irradiated and non-irradiated co-cultured cells. Concerning the signalling proteins, in this study, the attention was focussed on the release of the active and latent forms of the transforming growth factor-ß1 protein. Moreover, global gene expression profiles of non-transformed and transformed cells in untreated co-cultures were compared with those of 0.5-Gy-irradiated non-transformed cells co-cultured with the transformed cells. The results show an effect of radiation on the release of signalling proteins in the medium, although no significant differences in release rates were detectable when varying the doses in the range from 0.25 to 1 Gy. Moreover, gene expression results suggest an effect of radiation on both cell populations, pointing out specific signalling pathways that might be involved in the enhanced induction of apoptosis.

Investigation of radiation-induced multilayered signalling response of the inflammatory pathway.

Ionising radiation exposure of cells might induce the perturbation of cell functions and, in particular, the activation or inhibition of several important pathways. This perturbation can cause the deregulation of both intra- and extra-cellular signalling cascades (such as the inflammatory pathway) and alter not only the behaviour of directly exposed cells but also the neighbouring non-irradiated ones, through the so-called bystander effect. The aim of the present work was to investigate the complex nonlinear interactions between the inflammatory pathway and other strictly interlaced signalling pathways, such as Erk1/2 and Akt/PKB, focusing on the radiation-induced perturbation of such pathways in the dose range of 0-2 Gy. The results show how radiation affects these interconnected pathways and how confounding factors, such as the change of culture medium, can hide radiation-induced perturbations.

Radiosensitivity in lymphoblastoid cell lines derived from Shwachman-Diamond syndrome patients.

Shwachman-Diamond syndrome is an autosomal-recessive disorder characterised by bone marrow failure and a cumulative risk of progression to acute myeloid leukaemia. The Shwachman-Bodian-Diamond syndrome (SBDS) gene, the only gene known to be causative of the pathology, is involved in ribosomal biogenesis, stress responses and DNA repair, and the lack of SBDS sensitises cells to many stressors and leads to mitotic spindle destabilisation. The effect of ionising radiation on SBDS-deficient cells was investigated using immortalised lymphocytes from SDS patients in comparison with positive and negative controls in order to test whether, in response to ionising radiation exposure, any impairment in the DNA repair machinery could be observed. After irradiating cells with different doses of X-rays or gamma-rays, DNA repair kinetics and the residual damages using the alkaline COMET assay and the γ-H2AX assay were assessed, respectively. In this work, preliminary data about the comparison between ionising radiation effects in different patients-derived cells and healthy control cells are presented.

In vitro γ-ray-induced inflammatory response is dominated by culturing conditions rather than radiation exposures.

The inflammatory pathway has a pivotal role in regulating the fate and functions of cells after a wide range of stimuli, including ionizing radiation. However, the molecular mechanisms governing such responses have not been completely elucidated yet. In particular, the complex activation dynamics of the Nuclear transcription Factor kB (NF-kB), the key molecule governing the inflammatory pathway, still lacks a complete characterization. In this work we focused on the activation dynamics of the NF-kB (subunit p65) pathway following different stimuli. Quantitative measurements of NF-kB were performed and results interpreted within a systems theory approach, based on the negative feedback loop feature of this pathway. Time-series data of nuclear NF-kB concentration showed no evidence of γ-ray induced activation of the pathway for doses up to 5 Gy but highlighted important transient effects of common environmental stress (e.g. CO2, temperature) and laboratory procedures, e.g. replacing the culture medium, which dominate the in vitro inflammatory response.

Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.

Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.

Autoimmune bullous skin diseases occurring under anti-tumor necrosis factor therapy: two case reports.

BACKGROUND: Anti-tumor necrosis factor (TNF) agents are increasingly being used for a rapidly expanding number of rheumatic and systemic diseases. As a result of this use, and of the longer follow-up periods of treatment, there are a growing number of reports of the development of autoimmune processes related to anti-TNF agents. The use of anti-TNF agents has been associated with more and more cases of autoimmune diseases, principally cutaneous vasculitis, lupus-like syndrome, systemic lupus erythematosus and interstitial lung disease. OBSERVATIONS: We report 2 cases of autoimmune bullous skin disease occurring in patients undergoing TNF-targeted therapy: a bullous pemphigoid and a pemphigus foliaceus. Both patients were treated by anti-TNF agents for rheumatoid arthritis and showed improvement following interruption of that treatment. Here, we discuss the relationship between anti-TNF therapy and the occurrence of autoimmune bullous disease. CONCLUSION: Anti-TNF agents should be considered as a potential cause of drug-induced autoimmune bullous skin disease.

Lack of P-glycoprotein induction by rifampicin and phenobarbital in human lymphocytes.

The efficacy of drugs acting within lymphocytes depends on their intracellular concentrations, which could be modulated by membrane efflux transporters including P-glycoprotein (P-gp), encoded by the MDR1 gene. In particular, P-gp induction may compromise the efficacy of its substrates. Rifampicin and phenobarbital have been shown to induce P-gp in hepatic and intestinal cells through the activation of the nuclear receptors PXR and CAR. However, controversial data exist in human lymphocytes. We investigated the effect of these drugs on P-gp activity and expression in lymphocytes in vitro and ex vivo. CCRF-CEM cells and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were incubated in the presence of rifampicin, phenobarbital, or without any drug. P-gp activity was measured by flow cytometry using DiOC(6) efflux. MDR1, PXR and CAR mRNA expression were measured by quantitative RT-PCR. Neither P-gp activity nor MDR1 mRNA expression were modified by rifampicin or phenobarbital both in CCRF-CEM cells and PBMCs. Moreover, P-gp protein expression at the membrane was neither detectable nor induced. The very weak PXR and CAR mRNA expression levels in these cells could partly explain these results. Therefore, P-gp induction by rifampicin and phenobarbital may play a negligible role in drug interactions occurring within lymphocytes.

High levels of P-glycoprotein activity in human lymphocytes in the first 6 months of life.

P-glycoprotein (P-gp) is an efflux transporter that controls the intracellular concentrations of drugs. Human development may modulate P-gp function. We investigated the effect of age on P-gp activity and MDR1 gene expression in lymphocytes. We also assessed the influence of human immunodeficiency virus (HIV) infection. We used 3,3'-diethyloxacarbocyanin iodide (DiOC(6)) efflux, estimated by flow cytometry, to quantify P-gp activity in 94 children (age range, 0-18 years) and 25 adults. MDR1 gene expression was quantified using reverse transcription-PCR (RT-PCR). In T and natural killer (NK) cell populations, P-gp activity peaked at birth, decreased between the ages of 0 and 6 months, and stabilized between the ages of 6 months and 2 years (P < 10(-6)). These maturation profiles were also strongly correlated (r = 0.67, P < 10(-6)). HIV infection did not affect P-gp activity in the lymphocytes of children. MDR1 gene expression was not influenced by age, nor was it correlated with P-gp activity. The high levels of P-gp activity observed in the lymphocytes of children ~6 months of age may affect the efficacy of intracellular drugs.

Remission from Kaposi's sarcoma on HAART is associated with suppression of HIV replication and is independent of protease inhibitor therapy.

Highly active antiretroviral therapy (HAART) reduces the incidence and improves the prognosis of Kaposi's sarcoma (KS). This study was designed to identify factors associated with KS clinical responses in HIV-infected patients during HAART. We reviewed the files of 138 HIV-1-infected patients with KS. Epidemiologic and HIV-related clinical and biological parameters were recorded at KS diagnosis (baseline) and every 6 months thereafter. In a subset of 73 antiretroviral-naive patients, we compared the clinical outcome of KS according to the use or nonuse of protease inhibitors (PI). After 6 months of follow-up, KS remission was more frequent in patients who were naive of HAART and who were at ACTG stage S0 at baseline (P = 0.03 and 0.02). Undetectable HIV viral load was strongly associated with KS remission (P< or = 0.004 at all time points), while CD4 cell count was not. Among the 73 antiretroviral-naive patients at baseline, and who were studied for 24 months, KS outcome did not differ between patients who were prescribed PI-containing and PI-sparing regimens. Intercurrent multicentric Castleman's disease was associated with poor outcome after 60 months of follow-up (P< or = 0.0001). Fourteen deaths occurred after a median follow-up of 37.5 months, eight of which were KS related. Suppression of HIV replication appears to be crucial to control KS. Non-PI-based regimens were equivalent to PI-based regimens as regards the clinical and virological outcome of antiretroviral-naive HIV-infected patients with KS.

Electrocardiographic alterations in chronically Trypanosoma cruzi-infected persons exposed to cardiovascular factors.

METHODS: We analyzed the potential influence that associated risk factors (ARF), such as smoking, alcoholism, overweight, and hypertension, could have on the establishment of chronic chagasic cardiomyopathy (CC). The sample was comprised of 124 individuals, 69 males and 55 females (mean age +/- SD, 41 +/- 9.5 years), who were born in en demic areas of Northern Argentina and migrated further to Rosario City, an area where autochthonous cases of Chagas' disease have never been registered. Assessments included the following: clinical examination to discard previous cardiomyopathies; search for the presence of ARF according to standard criteria; specific serology; frontal chest X-ray, and 12-lead resting electrocardiogram (ECG). Subjects were classified on the basis of their serological status and presence of ARF into four groups: Tc+ARF+ T. cruzi-infected persons with ARF (n = 41); Tc-ARF+ seronegativity in presence of ARF (n = 27); Tc+ARF- individuals showing positive serology that lacked ARF (n = 27), and Tc-ARF- seronegative individuals having no ARF (n = 29). RESULTS: Except for a higher female/male ratio in groups presenting no ARF (p < 0.02), no statistical differences as to age, length of residence in endemicity areas (LR), and ARF distribution were recorded among groups. Forty-one persons presented abnormal ECG tracings, distributed thus: Tc+ARF+, 18/41; Tc-ARF+, 14/27, Tc+ARF-, 14/27, and Tc-ARF, 4/29 (p < 0.01, in relation to the latter group). Subjects from the Tc+ARF+, Tc-ARF+, and Tc+ARF- groups had 4.89-, 6.7-, and 6.7-fold increases, respectively, if having an abnormal ECG when compared with Tc-ARF- individuals. Comparisons on the frequency of abnormal ECG between seropositives carrying ARF or not yielded a non-significant odds ratio, be it estimated as crude, or after adjusting for sex, age, and LR in multivariate analysis. CONCLUSIONS: Presence of ARF was not associated with an increasing risk of cardiac affectation in chronically T. cruzi-infected persons, but resulted in chagasic-compatible ECG abnormalities in those seronegative individuals.

Psoriasis patients have T-cells with reduced responsiveness to common mycobacterial antigens.

Heparinised blood samples were obtained from 20 patients with chronic plaque psoriasis and from 13 age-matched healthy controls. After preliminary titration, mononuclear cells separated over Ficoll-Tryoson were cultured for 5 days with 10 microg ml(-1) of 15 mycobacterial preparations, or with pokeweed mitogen and concanavalin A. Stimulation indices were determined for each reagent and means were determined for patients and controls. Results for patients showed a striking reduction of responsiveness to mycobacteria, apparently due to loss of responses to group i, common mycobacterial antigens, and no differences in responses to mitogens. These observations relate psoriasis to certain other diseases, notably mycobacterial infections, rheumatoid arthritis, Chagas' disease and human immunodeficiency virus infection. The observations may be relevant to the aetiology of psoriasis, and to potential immunotherapy for the disease.

Impaired in vitro T-cell responses in patients with community acquired pneumonia.

To evaluate the status of the cellular immune response of patients with community acquired pneumonia (CAP), 8 CAP cases were studied for their in vitro T-cell responses to concanavalin A (Con A), tuberculin, and candidin, as well as levels of major T-cell populations in peripheral blood. Assessment on admission revealed that CAP patients had significantly decreased responses to both antigen and mitogen driven lymphocyte proliferation when compared to age and sex matched controls. Studies performed upon 1 week of antibiotic treatment made evident, in turn, that clinical improvement was accompanied by a reestablishment of the in vitro responses to tuberculin and candidin, whereas the lymphoproliferation induced by Con A remained decreased as in its first evaluation. Data from admission and day 7 of treatment showed no significant differences as to the levels of peripheral T-cell subsets when compared to those of healthy controls. Our results indicate that CAP coincides with reduced in vitro T-cell responses to antigen and mitogen stimulation.

Cellular immune response to common mycobacterial antigens in subjects seropositive for Trypanosoma cruzi.

The immune response is impaired in the silent stage of Chagas' disease. We used quadruple skin-testing with new tuberculins in 37 adults who were symptom-free but seropositive for Trypanosoma cruzi and in 37 matched seronegative controls. Whereas 19% of controls responded to common mycobacterial antigens, none of the Chagas' seropositive group responded to them (p < 0.006), demonstrating specificity in their unresponsiveness. The enhanced tuberculin reactivity after BCG vaccination in the control group was suppressed in seropositive subjects (p < 0.002). Selective loss of response to common mycobacterial antigens may have implications for the autoimmune pathology of Chagas' disease, and for susceptibility to tuberculosis, leprosy, and HIV disease.

Electrocardiographic alteration among first degree relatives with serologic evidence of Trypanosoma cruzi infection. A sibship study.

To analyze whether electrocardiographic alterations (ECGA) in patients with antibodies to Trypanosoma cruzi showed a pattern of familial aggregation, a sample of 379 young adults (166 men and 213 women) distributed in sibships, were assessed for the presence of anti-T. cruzi antibodies, and subjected to a complete clinical examination and a standard resting electrocardiogram (ECG). Positive T. cruzi serology was detected in 165 individuals, 48 of them showing an abnormal ECG (overall prevalence 29%). One hundred and eleven seropositive individuals were distributed in 45 sibships, each of them constituted by more than one seropositive sib, with ECGA being present in 34 out of these patients. Seropositive subjects with ECGA were detected in 27 sibships. Since the index case within each sibship is counted exactly once, affected individuals selected at random as propositi were extracted to calculate the prevalence of ECGA among first degree relatives of probands. Abnormal ECGs were recorded in 7 out of 45 sibs yielding a prevalence that did not differ from estimations registered in the general population or seropositive sibs. Data from the present sample show no familial aggregation for the occurrence of ECGA in patients with T. cruzi. infection.