Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies

OBJECTIVES: Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). METHODS: For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. RESULTS: Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients. CONCLUSION: These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA

Tunability and Losses of Mid-infrared Plasmonics in Heavily Doped Germanium Thin Films

Heavily-doped semiconductor films are very promising for application in mid-infrared plasmonic devices because the real part of their dielectric function is negative and broadly tunable in this wavelength range. In this work we investigate heavily n-type doped germanium epilayers grown on different substrates, in-situ doped in the $10^{17}$ to $10^{19}$ cm$^{-3}$ range, by infrared spectroscopy, first principle calculations, pump-probe spectroscopy and dc transport measurements to determine the relation between plasma edge and carrier density and to quantify mid-infrared plasmon losses. We demonstrate that the unscreened plasma frequency can be tuned in the 400 - 4800 cm$^{-1}$ range and that the average electron scattering rate, dominated by scattering with optical phonons and charged impurities, increases almost linearly with frequency. We also found weak dependence of losses and tunability on the crystal defect density, on the inactivated dopant density and on the temperature down to 10 K. In films where the plasma was optically activated by pumping in the near-infrared, we found weak but significant dependence of relaxation times on the static doping level of the film. Our results suggest that plasmon decay times in the several-picosecond range can be obtained in n-type germanium thin films grown on silicon substrates hence allowing for underdamped mid-infrared plasma oscillations at room temperature.Comment: 18 pages, 10 figure

The diagnostic role of total-body 18F-FDG PET/CT in patients with multiple tumors: a report of the association of thyroid cancer with lung or renal tumors

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Increased HMGB1 expression and release by mononuclear cells following surgical/anesthesia trauma

Introduction: High mobility group box 1 (HMGB1) is a key mediator of inflammation that is actively secreted by macrophages and/or passively released from damaged cells. The proinflammatory role of HMGB1 has been demonstrated in both animal models and humans, since the severity of inflammatory response is strictly related to serum HMGB1 levels in patients suffering from traumatic insult, including operative trauma. This study was undertaken to investigate HMGB1 production kinetics in patients undergoing major elective surgery and to address how circulating mononuclear cells are implicated in this setting. Moreover, we explored the possible relationship between HMGB1 and the proinflammatory cytokine interleukin-6 (IL-6). Methods: Forty-seven subjects, American Society of Anesthesiologists physical status I and II, scheduled for major abdominal procedures, were enrolled. After intravenous medication with midazolam (0.025 mg/Kg), all patients received a standard general anesthesia protocol, by thiopentone sodium (5 mg/Kg) and fentanyl (1.4 mu g/Kg), plus injected Vecuronium (0.08 mg/Kg). Venous peripheral blood was drawn from patients at three different times, t(0): before surgery, t(1): immediately after surgical procedure; t(2): at 24 hours following intervention. Monocytes were purified by incubation with anti-CD14-coated microbeads, followed by sorting with a magnetic device. Cellular localization of HMGB1 was investigated by flow cytometry assay; HMGB1 release in the serum by Western blot. Serum samples were tested for IL-6 levels by ELISA. A one-way repeated-measures analysis ANOVA was performed to assess differences in HMGB1 concentration over time, in monocytes and serum. Results: We show that: a) cellular expression of HMGB1 in monocytes at t(1) was significantly higher as compared to t(0); b) at t(2), a significant increase of HMGB1 levels was found in the sera of patients. Such an increase was concomitant to a significant down-regulation of cellular HMGB1, suggesting that the release of HMGB1 might partially derive from mononuclear cells; c) treatment of monocytes with HMGB1 induced in vitro the release of IL-6; d) at t(2), high amounts of circulating IL-6 were detected as compared to t(0). Conclusions: This study demonstrates for the first time that surgical/anesthesia trauma is able to induce an early intracellular upregulation of HMGB1 in monocytes of surgical patients, suggesting that HMGB1 derives, at least partially, from monocytes

Whole-body radioiodine effective half-life in patients with differentiated thyroid cancer

Background: Radioactive 131I (RAI) therapy is used in patients with differentiated thyroid cancer (DTC) after total thyroidectomy for remnant ablation, adjuvant treatment or treatment of persistent disease. 131I retention data, which are used to indicate the time at which a 131I treated DTC patient can be released from the hospital, may bring some insights regarding clinical factors that prolong the length of hospitalization. The aim of this study was to investigate the 131I whole-body retention in DTC patients during 131I therapy. Methods: We monitored 166 DTC patients to follow the 131I whole-body retention during 131I therapy with a radioactivity detector fixed on the ceiling of each protected room. A linear regression fit permitted us to estimate the whole-body 131I effective half-life in each patient, and a relationship was sought between patients’ clinical characteristics and whole-body effective 131I half-life. Results: The effective 131I half-life ranged from 4.08 to 56.4 h. At multivariable analysis, longer effective 131I half-life was related to older age and extensive extra-thyroid disease. Conclusions: 131I effective half-life during 131I treatment in DTC patients is highly variable among patients and is significantly longer in older and in patients with RAI uptake in large thyroid remnants or in extrathyroidal disease that significantly prolongs the whole-body retention of 131I

Initial Testing of an Approximated, Fast Calculation Procedure for Personalized Dosimetry in Radionuclide Therapy Based on Planar Whole-Body Scan and Monte-Carlo Specific Dose Rates from the OpenDose Project

Individualized dosimetry in nuclear medicine is currently at least advisable in order to obtain the best risk–benefit balance in terms of the maximal dose to lesions and under-threshold doses to radiosensitive organs. This article aims to propose a procedure for fast dosimetric calculations based on planar whole-body scintigraphy (WBS) images and developed to be employed in everyday clinical practice. Methods: For simplicity and legacy reasons, the method is based on planar imaging dosimetry, complemented with some assumptions on the radiopharmaceutical kinetics empirically derived from single-photon emission tomography/computed tomography (SPECT/CT) image analysis. The idea is to exploit a rough estimate of the time-integrated activity as has been suggested for SPECT/CT dosimetry but using planar images. The resulting further reduction in dose estimation accuracy is moderated by the use of a high-precision Monte-Carlo S-factor, such as those available within the OpenDose project. Results: We moved the problem of individualized dosimetry to a transformed space where comparing doses was imparted to the ICRP Average Male/Female computational phantom, resulting from an activity distribution related to patient’s pharmaceutical uptake. This is a fast method for the personalized dosimetric evaluation of radionuclide therapy, bearing in mind that the resulting doses are meaningful in comparison with thresholds calculated in the same framework. Conclusion: The simplified scheme proposed here can help the community, or even the single physician, establish a quantitative guide-for-the-eye approach to individualized dosimetry

Expression of Cellular and Extracellular TERRA, TERC and TERT in Hepatocellular Carcinoma

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC

Ablation rate after radioactive iodine therapy in patients with differentiated thyroid cancer at intermediate or high risk of recurrence: a systematic review and a meta-analysis

Purpose We performed a systematic review and a meta-analysis to investigate the successful ablation rate after radioiodine (RAI) administration in patients with differentiated thyroid cancer (DTC) at intermediate-high risk of recurrence. Methods: A comprehensive literature search of the PubMed, Scopus, and Web of Science databases was conducted according to the PRISMA statement. Results: The final analysis included 9 studies accounting for 3103 patients at intermediate-high risk of recurrence. In these patients, the successful ablation rates ranged from 51 to 94% with a 71% pooled successful ablation and were higher in intermediate (72%) than in high (52%)-risk patients. Despite the rigorous inclusion standards, a significant heterogeneity among the evaluated studies was observed. Higher administered RAI activities are associated with a lower successful ablation rate in the whole population and in the subgroup of high-risk patients. Furthermore, pooled recurrence rate in intermediate-risk patients achieving successful ablation was only 2% during the subsequent 6.4-year follow-up while the pooled recurrence rate was 14% in patients who did not achieve a successful ablation. Conclusion: In a large sample of 3103 patients at intermediate-high risk of persistent/recurrent disease, 71% of patients achieved a successful ablation. In these intermediate-risk patients, the probability of subsequent recurrence is low and most recurrence occurred in those with already abnormal findings at the first control

Antiphospholipid antibodies in patients with stroke during COVID-19: A role in the signaling pathway leading to platelet activation

Background: Several viral and bacterial infections, including COVID-19, may lead to both thrombotic and hemorrhagic complications. Previously, it has been demonstrated an "in vitro " pathogenic effect of "antiphospholipid " antibodies (aPLs), which are able to activate a proinflammatory and procoagulant phenotype in monocytes, endothelial cells and platelets. This study analyzed the occurrence of aPL IgG in patients with acute ischemic stroke (AIS) during COVID-19, evaluating the effect of Ig fractions from these patients on signaling and functional activation of platelets. Materials and methods: Sera from 10 patients with AIS during COVID-19, 10 non-COVID-19 stroke patients, 20 COVID-19 and 30 healthy donors (HD) were analyzed for anti-cardiolipin, anti-beta 2-GPI, anti-phosphatidylserine/prothrombin and anti-vimentin/CL antibodies by ELISA. Platelets from healthy donors were incubated with Ig fractions from these patients or with polyclonal anti-beta 2-GPI IgG and analyzed for phospho-ERK and phospho-p38 by western blot. Platelet secretion by ATP release dosage was also evaluated. Results: We demonstrated the presence of aPLs IgG in sera of patients with AIS during COVID-19. Treatment with the Ig fractions from these patients or with polyclonal anti-beta 2-GPI IgG induced a significant increase of phospho-ERK and phospho-p38 expression. In the same vein, platelet activation was supported by the increase of adenyl nucleotides release induced by Ig fractions. Conclusions: This study demonstrates the presence of aPLs in a subgroup of COVID-19 patients who presented AIS, suggesting a role in the mechanisms contributing to hypercoagulable state in these patients. Detecting these antibodies as a serological marker to check and monitor COVID-19 may contribute to improve the risk stratification of thromboembolic manifestations in these patients