Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto\u2019s thyroiditis

Abstract Background: Due to their \u201cnatural immune privilege\u201d and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. Hashimoto\u2019s thyroiditis (HT) is the most common thyroid autoimmune disease and cause of hypothyroidism. To date, conventional hormone replacement therapy and unspecific immunosuppressive regimens cannot provide a definitive cure for HT subjects. We explored the immunosuppressant potential of human f-LSCs on circulating lymphomonocytes (PBMCs) collected from healthy donors and female HT patients. Methods: We assessed the immunophenotyping of f-LSCs, both untreated and after 48 h of proinflammatory cytokine exposure, by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and flow cytometry. The immunosuppressant effects of f-LSCs on healthy activated PBMCs were investigated in cell-cell contact and transwell settings through cell cycle assay, acridine orange staining, and caspase-3 detection. We also studied T-cell responses and possible Treg conversion by means of flow cytometry. Functional assays were conducted in activated HT lymphocytes cocultured with f-LSCs after carboxyfluorescein succinimidyl ester labeling and intracellular detection of pro- and antiinflammatory cytokines. Results: The hypo-immunogenicity of the f-LSC population depended on both cell contact and soluble factors produced, as well as the undetectable expression of all those molecules required to fully activate T lymphocytes. Following exposure to Th1 cytokines, f-LSCs augmented expression of programmed death-ligand 1 and 2 (PDL-1 and -2), indoleamine-pyrrole-2,3-dioxygenase (IDO), interleukin (IL)-6, and monocyte chemotactic protein 1 (MCP-1) while maintaining their negative phenotype for major histocompatibility (MHC) class II and costimulatory molecules. During coculture, f-LSCs suppressed up to 40% of proliferation in healthy activated PBMCs, arrested them in the G0/G1 cell cycle phase without inducing apoptosis cascade, inverted the CD4/CD8 ratio, and promoted sustained expression of the immunomodulator marker CD69. Under coculture conditions the Th imbalance of autoreactive T cells from female HT patients was fully restored. Conclusions: Our study describes an in vitro coculture system able to prevent inappropriate activation of autoreactive T lymphocytes of female HT patients and to generate a tolerogenic environment even in an inflammatory background. Further investigations are necessary to establish whether this stem cell-based therapy approach in HT could avoid lifetime hormone replacement therapy by inducing T-cell education

MULTIPLE PLURIPOTENT STEM CELL MARKERS IN HUMAN ANAPLASTIC THYROID CANCER: THE PUTATIVE UPSTREAM ROLE OF SOX-2

Background: Anaplastic Thyroid Carcinoma (ATC) is a rare and aggressive endocrine tumor, with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were the following: 1) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers; 2) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch off. Methods: Ex vivo: eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by RT and qRT-PCR and immunohistochemistry. In vitro: in ATC SW1736 cells the expression levels of OCT-4, NANOG and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing. Results: OCT-4, KLF4 and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4 \ub1 2.1 % of cells were aldehyde dehydrogenase positive) and a high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, SSEA4). SOX2 silencing down-regulated OCT-4, NANOG and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8 fold) in comparison to control cells with 10 \ub5M cisplatin (93.9\ub13.4% vs. 52.6\ub19.4%, p<0.01) and 2.7 fold with 0.5\ub5M doxorubicin (45.8\ub19.9% vs. 17.1\ub13.4% p<0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9\ub11.4%) and doxorubicin treatment (74.1\ub10.1%) vs. no-target-treated cells (respectively, 45.8\ub11.0% and 48.6\ub11.0%, p<0.001). Conclusions: The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance

Radiobiological outcomes, microdosimetric evaluations and monte carlo predictions in eye proton therapy

CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) was the first Italian protontherapy facility dedicated to the treatment of ocular neoplastic pathologies. It is in operation at the LNS Laboratories of the Italian Institute for Nuclear Physics (INFN-LNS) and to date, 500 patients have been successfully treated. Even though proton therapy has demonstrated success in clinical settings, there is still a need for more accurate models because they are crucial for the estimation of clinically relevant RBE values. Since RBE can vary depending on several physical and biological parameters, there is a clear need for more experimental data to generate predictions. Establishing a database of cell survival experiments is therefore useful to accurately predict the effects of irradiations on both cancerous and normal tissue. The main aim of this work was to compare RBE values obtained from in-vitro experimental data with predictions made by the LEM II (Local Effect Model), Monte Carlo approaches, and semi-empirical models based on LET experimental measurements. For this purpose, the 92.1 uveal melanoma and ARPE-19 cells derived from normal retinal pigmented epithelium were selected and irradiated in the middle of clinical SOBP of the CATANA proton therapy facility. The remarkable results show the potentiality of using microdosimetric spectrum, Monte Carlo simulations and LEM model to predict not only the RBE but also the survival curves

In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

Background: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. Methodology/Principal Findings: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133pos cells only in ARO and KAT-4 (6469% and 57612%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/ CD133pos cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133neg. Furthermore, ARO/CD133pos showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133neg was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133pos in comparison to ARO/CD133neg cells. The stem cell markers c- KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133pos when compared with ARO/CD133neg cells. Conclusions/Significance: We describe CD133pos cells in ATC cell lines. ARO/CD133pos cells exhibit stem cell-like features - such as high proliferation, self-renewal ability, expression of OCT-4 - and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches

Shattered pellet injection experiments at JET in support of the ITER disruption mitigation system design

A series of experiments have been executed at JET to assess the efficacy of the newly installed shattered pellet injection (SPI) system in mitigating the effects of disruptions. Issues, important for the ITER disruption mitigation system, such as thermal load mitigation, avoidance of runaway electron (RE) formation, radiation asymmetries during thermal quench mitigation, electromagnetic load control and RE energy dissipation have been addressed over a large parameter range. The efficiency of the mitigation has been examined for the various SPI injection strategies. The paper summarises the results from these JET SPI experiments and discusses their implications for the ITER disruption mitigation scheme

Interpretative and predictive modelling of Joint European Torus collisionality scans

Transport modelling of Joint European Torus (JET) dimensionless collisionality scaling experiments in various operational scenarios is presented. Interpretative simulations at a fixed radial position are combined with predictive JETTO simulations of temperatures and densities, using the TGLF transport model. The model includes electromagnetic effects and collisions as well as □(→┬E ) X □(→┬B ) shear in Miller geometry. Focus is on particle transport and the role of the neutral beam injection (NBI) particle source for the density peaking. The experimental 3-point collisionality scans include L-mode, and H-mode (D and H and higher beta D plasma) plasmas in a total of 12 discharges. Experimental results presented in (Tala et al 2017 44th EPS Conf.) indicate that for the H-mode scans, the NBI particle source plays an important role for the density peaking, whereas for the L-mode scan, the influence of the particle source is small. In general, both the interpretative and predictive transport simulations support the experimental conclusions on the role of the NBI particle source for the 12 JET discharges

Overview of JET results for optimising ITER operation

The JET 2019–2020 scientific and technological programme exploited the results of years of concerted scientific and engineering work, including the ITER-like wall (ILW: Be wall and W divertor) installed in 2010, improved diagnostic capabilities now fully available, a major neutral beam injection upgrade providing record power in 2019–2020, and tested the technical and procedural preparation for safe operation with tritium. Research along three complementary axes yielded a wealth of new results. Firstly, the JET plasma programme delivered scenarios suitable for high fusion power and alpha particle (α) physics in the coming D–T campaign (DTE2), with record sustained neutron rates, as well as plasmas for clarifying the impact of isotope mass on plasma core, edge and plasma-wall interactions, and for ITER pre-fusion power operation. The efficacy of the newly installed shattered pellet injector for mitigating disruption forces and runaway electrons was demonstrated. Secondly, research on the consequences of long-term exposure to JET-ILW plasma was completed, with emphasis on wall damage and fuel retention, and with analyses of wall materials and dust particles that will help validate assumptions and codes for design and operation of ITER and DEMO. Thirdly, the nuclear technology programme aiming to deliver maximum technological return from operations in D, T and D–T benefited from the highest D–D neutron yield in years, securing results for validating radiation transport and activation codes, and nuclear data for ITER

Testing a prediction model for the H-mode density pedestal against JET-ILW pedestals

The neutral ionisation model proposed by Groebner et al (2002 Phys. Plasmas 9 2134) to determine the plasma density profile in the H-mode pedestal, is extended to include charge exchange processes in the pedestal stimulated by the ideas of Mahdavi et al (2003 Phys. Plasmas 10 3984). The model is then tested against JET H-mode pedestal data, both in a 'standalone' version using experimental temperature profiles and also by incorporating it in the Europed version of EPED. The model is able to predict the density pedestal over a wide range of conditions with good accuracy. It is also able to predict the experimentally observed isotope effect on the density pedestal that eludes simpler neutral ionization models

New H-mode regimes with small ELMs and high thermal confinement in the Joint European Torus

New H-mode regimes with high confinement, low core impurity accumulation, and small edge-localized mode perturbations have been obtained in magnetically confined plasmas at the Joint European Torus tokamak. Such regimes are achieved by means of optimized particle fueling conditions at high input power, current, and magnetic field, which lead to a self-organized state with a strong increase in rotation and ion temperature and a decrease in the edge density. An interplay between core and edge plasma regions leads to reduced turbulence levels and outward impurity convection. These results pave the way to an attractive alternative to the standard plasmas considered for fusion energy generation in a tokamak with a metallic wall environment such as the ones expected in ITER.&amp; nbsp;Published under an exclusive license by AIP Publishing

The role of ETG modes in JET-ILW pedestals with varying levels of power and fuelling

We present the results of GENE gyrokinetic calculations based on a series of JET-ITER-like-wall (ILW) type I ELMy H-mode discharges operating with similar experimental inputs but at different levels of power and gas fuelling. We show that turbulence due to electron-temperature-gradient (ETGs) modes produces a significant amount of heat flux in four JET-ILW discharges, and, when combined with neoclassical simulations, is able to reproduce the experimental heat flux for the two low gas pulses. The simulations plausibly reproduce the high-gas heat fluxes as well, although power balance analysis is complicated by short ELM cycles. By independently varying the normalised temperature gradients (omega(T)(e)) and normalised density gradients (omega(ne )) around their experimental values, we demonstrate that it is the ratio of these two quantities eta(e) = omega(Te)/omega(ne) that determines the location of the peak in the ETG growth rate and heat flux spectra. The heat flux increases rapidly as eta(e) increases above the experimental point, suggesting that ETGs limit the temperature gradient in these pulses. When quantities are normalised using the minor radius, only increases in omega(Te) produce appreciable increases in the ETG growth rates, as well as the largest increases in turbulent heat flux which follow scalings similar to that of critical balance theory. However, when the heat flux is normalised to the electron gyro-Bohm heat flux using the temperature gradient scale length L-Te, it follows a linear trend in correspondence with previous work by different authors