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

In vitro generation of pancreatic endocrine cells from human adult fibroblast-like limbal stem cells

Stem cells might provide unlimited supply of transplantable cells for β-cell replacement therapy in diabetes. The human limbus is a highly specialized region hosting a well-recognized population of epithelial stem cells, which sustain the continuous renewal of the cornea, and the recently identified stromal fibroblast-like stem cells (f-LSCs), with apparent broader plasticity. However, the lack of specific molecular markers for the identification of the multipotent limbal subpopulation has so far limited the investigation of their differentiation potential. In this study we show that the human limbus contains uncommitted cells that could be potentially harnessed for the treatment of diabetes. Fourteen limbal biopsies were obtained from patients undergoing surgery for ocular diseases not involving the conjunctiva or corneal surface. We identified a subpopulation of f-LSCs characterized by robust proliferative capacity, expressing several pluripotent stem cell markers and exhibiting self-renewal ability. We then demonstrated the potential of f-LSCs to differentiate in vitro into functional insulin-secreting cells by developing a four-step differentiation protocol that efficiently directed f-LSCs towards the pancreatic endocrine cell fate. The expression of specific endodermal, pancreatic, islet, and β-cell markers, as well as functional properties of f-LSC-derived insulin-producing cells, were evaluated during differentiation. With our stage-specific approach, up to 77% of f-LSCs eventually differentiated into cells expressing insulin (also assessed as C-peptide) and exhibited phenotypic features of mature β-cells, such as expression of critical transcription factors and presence of secretory granules. Although insulin content was about 160-fold lower than what observed in adult islets, differentiated cells processed ∼98% of their proinsulin content, similar to mature β-cells. Moreover, they responded in vitro in a regulated manner to multiple secretory stimuli, including glucose. In conclusion, f-LSCs represent a possible relevant source of autologous, transplantable, insulin-producing cells that could be tested for the reversal of diabetes

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

Hepatic incidentaloma: An asymptomatic ectopic thyroid tissue

: An ectopic thyroid is a form of thyroid dysgenesis in which the entire thyroid gland or parts of it may be located in another part of the body than the usual place. The most frequent location is the base of the tongue. Although most cases are asymptomatic, symptoms related to tumor size and its relationship with surrounding tissues, hormonal dysfunction, and seldom malignancy may also occur. Here, we describe the case of an asymptomatic woman who was thyroidectomized 19 years previously for a toxic goiter and treated with conventional L-thyroxine therapy, until we enacted a progressive reduction of dosage of the replacement therapy. Incidentally, because of occasional abdomen discomfort, she was hospitalized in our Division of Endocrinology as there was ultrasound evidence of a large mass in the liver dislocating and imprinting the choledochal duct in the pre-pancreatic site, the gallbladder, and the cystic duct, which could not be dissociated from the contiguous hepatic parenchyma and was in very close proximity to the second duodenal portion and the head of the pancreas. Imaging techniques, such as TC, MR, TC/PET, and 131I scintigraphy, confirmed the large lesion with a diameter on the axial plane of about 8 × 5.5 cm and a cranio-caudal extension of about 6 cm. The impossibility of surgical debulking and/or radiometabolic 131I therapy, in the absence of compression symptoms, led to the multidisciplinary decision of a clinical and instrumental follow-up of this rare lesion

The integrated engineering design concept of the upper limiter within the EU-DEMO LIMITER system

The EU-DEMO first wall protection relies on a system of limiters. Although they are primarily designed for facing the energy released by a limited plasma during transients, their design should safely withstand a combination of loads relevant for in-vessel components (IVCs) during steady-state operation. They are not meant to breed tritium, nor to provide plasma stability. However, sitting in place of blanket portions, they should ensure an adequate shielding function to vacuum vessel and magnets while withstanding both their dead weight and the electro-mechanical loads arising from the interaction between current induced in the conductive structure and magnetic field. During plasma disruptions they will be subjected to halo currents flowing from/to the plasma and the grounded structures, whose effects must be added to the eddy current ones. Disruption-induced electro-mechanical loads are hence IVC design-driving, despite the uncertainties in both eddy and halo currents' magnitude and distribution, which depend on IVC design, electrical connectivity, plasma temperature and halo width. The integrated design of the limiter is made of two actively water-cooled sub-components: the Plasma-Facing Wall (PFW) directly exposed to the plasma, and the Shielding Block (SB) devoted to hold the PFW while providing neutronic shielding. The PFW design is driven by disruptive heat loads. Disruption-induced electro-magnetic loads are instead SB design drivers, meaning that the design details (i.e. geometry, electrical connections, attachments) affect the loads acting on it, which, in turn, are affected by the mechanical response of the structure. The present paper describes the design workflow and assessment of the Upper Limiter (UL), resulting from a close and iterative synergy among different fields. Built on static-structural and energy balance hand calculations based on, respectively, preliminary electro-magnetic and neutronic loads, the UL integrated design performance has then been verified against electro-magnetic, neutronic, thermal-hydraulic and structural assessment under the above-mentioned load combination. The outcome will be taken as reference for future limiter engineering designs

The integrated engineering design concept of the upper limiter within the EU-DEMO LIMITER system

The EU-DEMO first wall protection relies on a system of limiters. Although they are primarily designed for facing the energy released by a limited plasma during transients, their design should safely withstand a combination of loads relevant for in-vessel components (IVCs) during steady-state operation. They are not meant to breed tritium, nor to provide plasma stability. However, sitting in place of blanket portions, they should ensure an adequate shielding function to vacuum vessel and magnets while withstanding both their dead weight and the electro-mechanical loads arising from the interaction between current induced in the conductive structure and magnetic field. During plasma disruptions they will be subjected to halo currents flowing from/to the plasma and the grounded structures, whose effects must be added to the eddy current ones. Disruption-induced electro-mechanical loads are hence IVC design-driving, despite the uncertainties in both eddy and halo currents’ magnitude and distribution, which depend on IVC design, electrical connectivity, plasma temperature and halo width. The integrated design of the limiter is made of two actively water-cooled sub-components: the Plasma-Facing Wall (PFW) directly exposed to the plasma, and the Shielding Block (SB) devoted to hold the PFW while providing neutronic shielding. The PFW design is driven by disruptive heat loads. Disruption-induced electro-magnetic loads are instead SB design drivers, meaning that the design details (i.e. geometry, electrical connections, attachments) affect the loads acting on it, which, in turn, are affected by the mechanical response of the structure. The present paper describes the design workflow and assessment of the Upper Limiter (UL), resulting from a close and iterative synergy among different fields. Built on static-structural and energy balance hand calculations based on, respectively, preliminary electro-magnetic and neutronic loads, the UL integrated design performance has then been verified against electro-magnetic, neutronic, thermal-hydraulic and structural assessment under the above-mentioned load combination. The outcome will be taken as reference for future limiter engineering designs

Robustness of pet radiomics features: Impact of co-registration with mri

Radiomics holds great promise in the field of cancer management. However, the clinical application of radiomics has been hampered by uncertainty about the robustness of the features extracted from the images. Previous studies have reported that radiomics features are sensitive to changes in voxel size resampling and interpolation, image perturbation, or slice thickness. This study aims to observe the variability of positron emission tomography (PET) radiomics features under the impact of co-registration with magnetic resonance imaging (MRI) using the difference percentage coefficient, and the Spearman’s correlation coefficient for three groups of images: (i) original PET, (ii) PET after co-registration with T1-weighted MRI and (iii) PET after co-registration with FLAIR MRI. Specifically, seventeen patients with brain cancers undergoing [11C]-Methionine PET were considered. Successively, PET images were co-registered with MRI sequences and 107 features were extracted for each mentioned group of images. The variability analysis revealed that shape features, first-order features and two subgroups of higher-order features possessed a good robustness, unlike the remaining groups of features, which showed large differences in the difference percentage coeffi-cient. Furthermore, using the Spearman’s correlation coefficient, approximately 40% of the selected features differed from the three mentioned groups of images. This is an important consideration for users conducting radiomics studies with image co-registration constraints to avoid errors in cancer diagnosis, prognosis, and clinical outcome prediction

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