Neutron Autoradiography Combined With UV-C Sensitization: Toward the Intracellular Localization of Boron

Our group has reported the imprint formation of biological material on polycarbonate nuclear track detectors by UV-C exposure, which is used as an approach to simultaneously visualize cell imprints and nuclear tracks coming from the boron neutron capture reaction. Considering that the cell nucleus has a higher UV-C absorption than the cytoplasm and that hematoxylin preferentially stains the nucleus, we proposed to enhance the contrast between these two main cell structures by hematoxylin staining before UV-C sensitization. In this study, several experiments were performed in order to optimize UV-C exposure parameters and chemical etching conditions for cell imprint formation using the SK-BR-3 breast cancer cell line. The proposed method improves significantly the resolution of the cell imprints. It allows clear differentiation of the nucleus from the rest of the cell, together with nuclear tracks pits. Moreover, it reduces considerably the UV-C exposure time, an important experimental issue. The proposed methodology can be applied to study the boron distribution independently from the chosen cell line and/or boron compounds.Fil: Gadan, Mario Alberto. Comisión Nacional de Energía Atómica; ArgentinaFil: Lloyd, Rodrigo. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Científica y Tecnológica; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Saint Martin, María Laura Gisela. Comisión Nacional de Energía Atómica; ArgentinaFil: Olivera, María S.. Comisión Nacional de Energía Atómica; ArgentinaFil: Policastro, Lucia Laura. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes | Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes.; ArgentinaFil: Portu, Agustina Mariana. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Nuclear Physics meets Medicine and Biology: Boron Neutron Capture Therapy

BNCT is a tumour treatment based on thermal-neutron irradiation of tissues enriched with 10B, which according to the 10B(n, )7Li reaction produces particles with high Linear Energy Transfer and short range. Since this treatment can deliver a therapeutic tumour dose sparing normal tissues, BNCT represents an alternative for diffuse tumours and metastases, which show poor response to surgery and photontherapy. In 2001 and 2003, in Pavia BNCT was applied to an isolated liver, which was infused with boron, explanted, irradiated and re-implanted. A new project was then initiated for lung tumours, developing a protocol for Boron concentration measurements and performing organ-dose Monte Carlo calculations; in parallel, radiobiology studies are ongoing to characterize the BNCT effects down to cellular level. After a brief introduction, herein we will present the main activities ongoing in Pavia including the radiobiological ones, which are under investigation not only experimentally but also theoretically, basing on a Monte Carlo code recently extended to simulate cell killing

Defunctioning stoma and short- and long-term outcomes after low anterior resection for rectal cancer : a nationwide register–based cohort study

Purpose: A defunctioning stoma reduces the risk of symptomatic anastomotic leakage after low anterior resection for rectal cancer and mitigates the consequences when a leakage occurs, but the impact on mortality and oncological outcomes is unclear. The aim was to investigate the associations of a defunctioning stoma with short- and long-term outcomes in patients undergoing low anterior resection for rectal cancer. Methods: Data from all patients who underwent curative low anterior resection for rectal cancer between 1995 and 2010 were obtained from the Swedish Colorectal Cancer Register. A total of 4130 patients, including 2563 with and 1567 without a defunctioning stoma, were studied. Flexible parametric models were used to estimate hazard ratios for all-cause mortality, 5-year local recurrence, and distant metastatic disease in relation to the use of defunctioning stoma, adjusting for confounding factors and accounting for potential time-dependent effects. Results: During a median follow-up of 8.3 years, a total of 2169 patients died. In multivariable analysis, a relative reduction in mortality was observed up to 6 months after surgery (hazard ratio = 0.82: 95% CI 0.67–0.99), but not thereafter. After 5 years of follow-up, 4.2% (173/4130) of the patients had a local recurrence registered and 17.9% (741/4130) had developed distant metastatic disease, without difference between patients with and without defunctioning stoma. Conclusion: A defunctioning stoma is associated with a short-term reduction in all-cause mortality in patients undergoing low anterior resection for rectal cancer without any difference in long-term mortality and oncological outcomes, and should be considered as standard of care

Testing of a gel equivalent to liver to perform neutron characterizations

Several characterizations are needed in the frame of the project of irradiation of sections II-III of the human liver left lobe. For trials including neutron irradiation, samples remain with a certain level of activity that avoids their promptly disposal. To not use biological materials, that degrades and add the requirement of proper conservation up to their safe disposal, gel phantoms with non-residual activation were developed and their behavior under neutron irradiation was studied and compared to the one already obtained in previous studies using a pig liver. Considering that response to neutrons depends mainly on the hydrogen content of the human tissue, materials chosen to construct phantoms equivalent to sections II-III of the liver left lobe were demineralized water and 2% agarose (C12H14O2(OH)4). The solution, still in its liquid phase, was poured into specially designed polyethylene bags in order to reproduce the portion of liver to be treated. To cover the variability range of sizes and weights in humans, three phantoms were prepared: 180, 240 and 300 g. Implantable rhodium based self-powered detectors were used to obtain neutron flux profiles in the developed phantoms, both external and internal. Implantation of SPND was done along the central longitudinal axis of the samples, where lowest flux is expected. Irradiations were carried out under the same conditions that are previewed for the liver, i.e. inside the same acrylic container and covered with demineralized water to simulate preservation solution. Phantoms construction was quite simple and its durability very good. Handling in order to insert instrumentation was easy. Obtained internal neutron profile resulted very similar to the obtained for the pig liver, which showed that it is possible to use the phantoms to perform neutron characterizations

Stress-Induced Reversion to Virulence of Infectious Pancreatic Necrosis Virus in Naïve Fry of Atlantic Salmon (<em>Salmo salar</em> L.)

<div><p>We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (<i>Salmo salar</i> L.) fry. Naïve fry were persistently infected with a virulent strain (T₂₁₇A₂₂₁ of major structural virus protein 2, VP2) or a low virulent (T₂₁₇T₂₂₁) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T₂₁₇T₂₂₁ infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T₂₁₇T₂₂₁ infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied <i>in vitro</i> under an antiviral state induced by recombinant IFN-α1. The T₂₁₇A₂₂₁ reverted variant replicated to levels 23-fold higher than the T₂₁₇T₂₂₁ strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an <i>in vivo</i> trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication <i>in vivo</i> and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.</p> </div

Chromatogram of TT-infected fish 1 and 14 days post stress.

<p>Details from chromatogram of TT-infected fish at 1 day (A) and 14 days (B) post stress. Area indicated is from the hypervariable region of VP2. The chromatograms show that amino acid positions 217, 221 and 247 remain unchanged. Further there are no double-peaks in codons of any of these residues.</p

The mRNA expression of IgM, RAG-1 and TCR.

<p>The mRNA expression at different sizes of Atlantic salmon fry is indicated, showing no expression at 0.15 g size with significant increase by 1.5 g for IgM and RAG-1 while TCR is significantly upregulated by 2 g. p<0.001, n = 6; One-way Anova.</p

Virus detection by cell culture and RT-PCR post stress.

<p>Number of fish found positive by cell culture (cc) and reverse transcriptase PCR (RT-PCR) at different time points after the stress period was completed for the TA and TT infected groups. Fish negative by cell culture were subsequently examined by RT-PCR. Results for non-stressed controls and stressed groups are given and show that stress results in higher number of virus positive fish by cell culture at 28 days post stress. N = 6 per group.</p