Jacopo Morini

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.

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 5Gy 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.

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-β1 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.

Parental origin of the deletion del(20q) in Shwachman-Diamond patients and loss of the paternally derived allele of the imprinted L3MBTL1 gene.

Shwachman–Diamond syndrome (SDS) (OMIM 260400) is a rare autosomal recessive disease characterized by exocrine pancreatic insufficiency, skeletal, and hematological abnormalities and bone marrow (BM) dysfunction. Mutations in the SBDS gene cause SDS. Clonal chromosome anomalies are often present in BM, i(7)(q10) and del(20q) being the most frequent ones. We collected 6 SDS cases with del(20q): a cluster of imprinted genes, including L3MBTL1 and SGK2 is present in the deleted region. Only the paternal allele is expressed for these genes. Based on these data, we made the hypothesis that the loss of this region, in relation to parental origin of deletion, may be of relevance for the hematological phenotype. By comparing hematological data of our 6 cases with a group of 20 SDS patients without evidence of del(20q) in BM, we observed a significant difference for Hb levels (P < 0.012), and a difference slightly above the significance level for RBC counts (P < 0.053): in both cases the values were higher in patients with del(20q). We also report preliminary evidence for an increased number of BFU‐E colonies in cases with paternal deletion, data on the presence of the deletion in colonies and in mature circulating lymphocytes.

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.

The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum

Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0–10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction scaffold protein expression levels, assumed to be related to IFN-γ- and TNF-α-mediated signaling.

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.

Absence of acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 in a series of 10 patients with Shwachman-Diamond syndrome

We report that acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 was not identified in a series of 10 patients with Shwachman–Diamond syndrome (SDS). Shwachman–Diamond syndrome (Online Mendelian Inheritance in Man reference 260400) is a rare autosomal recessive disease first reported in 1964 (Bodian et al,1964; Shwachman et al,1964) and characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/ AML). Almost 90% of SDS cases are caused by two common mutations, c. [183_184TA>CT] and c.[258 + 2T>C], in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent are an isochromosome for long arms of chromosome 7, i(7)(q10) and an interstitial deletion of long arms of chromosome 20, del(20)(q11) (Maserati et al, 2009). An earlier study analysed BM DNA of eight cases with the i(7)(q10) and demonstrated that all of them carried a double dose of the c.[258 + 2T>C] (Minelli et al, 2009). This result suggested that, as the c.[258 + 2T>C] mutation still allows the production of some amount of normal protein (Austin et al, 2005), this could contribute to the low incidence of MDS/AML observed in this subset of SDS patients. Recently, Parikh et al (2012) described acquired CN-LOH for most of 7q in a patient with SDS, also showing that the clone of BM cells with CN-LOH contained two copies of the gene with the c.[258 + 2T>C] mutation. The presence of the CN-LOH in BM cells mimics the presence of i(7)(q10): both mechanisms produce a duplication of the c.[258 + 2T>C] mutation, together with the probable associated positive effects. Parikh et al (2012) also outlined that the genetic variation of the CN-LOH could provide an explanation for clonal expansion of the affected haematopoietic progenitor cell. Consequently, we investigated if, to provide the cell with an extra copy of the c.[258 + 2T>C] mutation, this could be a common mechanism in addition to the typical i(7)(q10) frequently observed in BM from SDS patients. We collected BM samples from 10 SDS patients in whom the cytogenetic analysis demonstrated a normal karyotype (eight cases) or the presence of the 20q deletion (two cases). The genotypes were c.[258 + 2T>C]/[183_184TA>CT] in seven cases and c.[258 + 2T>C]/[others] in three cases. Morphological analysis of the BM aspirate, available for six patients, showed normal or hypoplastic cellularity in three, and normal or hyperplastic cellularity with minimal decrease of erythropoiesis, megakaryopoiesis and myelopoiesis in the remaining three. These data are in keeping with the karyotype-phenotype correlation discussed by Pressato et al (2012). Microsatellite analysis, performed as reported in Minelli et al (2009), demonstrated a normal dosage between paternal and maternal alleles, excluding the presence of uniparental disomy related to chromosome 7, in all BM samples examined (Table I and Fig 1A). The method is able to identify the presence of the chromosomal anomaly if it is present in at least 20% of cells (Minelli et al, 2009). To verify the reliability of the method, we sequenced exon 2 of the SBDS gene from the BM of two SDS patients with 50–70% cells with i(7)(q10) and, as expected, found that the mutant peak (C) was higher than the wild-type peak (T) (Fig 1B). Exon 2 of the SBDS gene was then sequenced using BM and peripheral blood samples and, in all cases analysed, the mutant peak (C) and the wild-type peak (T) for the c.[258 + 2T>C] mutation were exactly the same height in both samples (Fig 1C) whereas Parikh et al (2012) observed an higher peak for the mutated allele when BM and fibroblasts were compared. Thus, no evidence for CN-LOH was found in any of the patients studied. If the hypothesis that an increased level of the c.[258 + 2T>C] mutation on a SDS background (c.[183_184TA>CT]/c.[258 + 2T>C]) provides a selective advantage, we would expect to identify cases in whom a clone showing a (partial) loss of the c.[183_184TA>CT] or an homozygous c.[258 + 2T>C] is present.

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.

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.