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BackgroundInformation on the clinical characteristics and pathophysiological mechanisms underlying post-COVID-19 fatigue are scarce. The main objective of this study was to evaluate sex-specific humoral and T-cell responses associated with post-COVID-19 fatigue in a sample of individuals treated as outpatients.MethodsAt a median time of 279 (179;325) days after the acute infection, a total of 281 individuals (45.9% men) aged 18-87 years old were included in the analysis. The participants were examined at the University Hospital of Augsburg, Southern Germany. Fatigue was assessed using the Fatigue Assessment Scale (FAS). Levels of anti-SARS-CoV2-spike IgG antibodies were measured by an enzyme-linked immunosorbent assay (ELISA), and for exploration of the SARS-CoV2-specific T-cell response, ex vivo ELISpot/FLUOROspot assays were conducted using an interferon-γ (IFN-γ) and interleukin-2 (IL-2) SARS-CoV-iSpot kit.ResultsWomen more significantly suffered from post-COVID-19 fatigue in comparison to men (47.4% versus 25.6%, p=0.0002). Females but not males with fatigue showed a significantly lower number of T-cells producing IFN-γ, IL-2 or both IL-2 and IFNγ in comparison with females without fatigue. In both sexes, serum levels of anti-SARS-CoV2-spike IgG antibodies did not differ significantly between participants with or without fatigue.ConclusionsDevelopment of fatigue after acute COVID-19 disease might be associated with SARS-CoV-2-specific T-cell responses in women, but not men after a mild infection course treated outpatient.

Background Currently, more than 30,200,000 COVID-19 cases have been diagnosed in Germany alone. However, data regarding prevalence of COVID-19 in children, both in Germany and internationally, are sparse. We sought to evaluate the number of infected children by measuring IgG antibodies. Methods Oropharyngeal swabs were collected between December 2020 and August 2021 to measure SARS-CoV-2, and capillary blood for the detection of SARS-CoV-2 antibodies (by rapid test NADAL® and filter paper test Euroimmun® ELISA); venous blood was taken for validation (Roche® ECLIA and recomLine Blot) in 365 German children aged 3–16 years from 30 schools and preschools. We used multiple serological tests because the filter paper test Euroimmun® ELISA performs better in terms of sensitivity and specificity than the rapid test NADAL®. The Roche® ECLIA test is used to detect SARS-CoV-2 spike protein, and the recomLine Blot test is used to rule out the possibility of infection by seasonal SARS-viruses and to test for specific SARS-CoV-2 proteins (NP, RBD and S1). In addition, one parent each (n = 336), and 4–5 teachers/caregivers (n = 90) per institution were tested for IgG antibodies from capillary blood samples. The total study duration was 4 months per child, including the first follow-up after 2 months and the second after 4 months. Results Of 364 children tested at baseline, 3.6% (n = 13) were positive for SARS-CoV-2 IgG antibodies using Euroimmun® ELISA. Seven children reported previously testing positive for SARS-CoV-2; each of these was confirmed by the Roche® Anti-SARS-CoV-2-ECLIA (antibody to spike protein 1) test. SARS-CoV-2 IgG antibodies persisted over a 4-month period, but levels decreased significantly (p = 0.004) within this timeframe. The median IgG values were 192.0 BAU/ml [127.2; 288.2], 123.6 BAU/ml [76.6; 187.7] and 89.9 BAU/ml [57.4; 144.2] at baseline, 2 months and 4 months after baseline, respectively. During the study period, no child tested positive for SARS-CoV-2 by oropharyngeal swab. A total of 4.3% of all parents and 3.7% of teachers/caregivers tested positive for IgG antibodies by Euroimmun® ELISA at baseline. Conclusion We noted a rather low seroprevalence in children despite an under-reporting of SARS-CoV-2 infections. Measurement of IgG antibodies derived from capillary blood appears to be a valid tool to detect asymptomatic infections in children. However, no asymptomatic active infection was detected during the study period of 4 months in the whole cohort. Further data on SARS-CoV-2 infections in children are needed, especially in the group of

Context: Micro-RNA have emerged as an important class of short endogenous RNA that act as posttranscriptional regulators of gene expression and are constantly deregulated in human cancer. MiR-1 has been found down-regulated in lung, colon, and prostate cancer. Objectives: In this study, we investigated the possible role of miR-1 in thyroid carcinogenesis. Design: We have analyzed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions and searched for miR-1 targets. Results: Our results show that miR-1 expression is drastically down-regulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 down-regulation was also found in thyroid hyperproliferative nonneoplastic lesions such as goiters. We identified the CCND2, coding for the cyclin D2 (CCND2) protein that favors the G1/S transition, CXCR4, and SDF-1± genes, coding for the receptor for the stromal cell derived factor-1 (SDF-1)/CXCL12 chemokine and its ligand SDF-1/CXCL12, respectively, as miR-1 targets. An inverse correlation was found between miR-1 expression and CXC chemokine receptor 4 (CXCR4) and SDF-1± protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1± proteins in cell invasion and metastasis, functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration. Conclusions: These results indicate the involvement of miR-1 in thyroid cell proliferation and migration, validating a role of miR-1 down-regulation in thyroid carcinogenesis.

Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype. [Cancer Res 2009;69(17):7079–87]

PGE2plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.

We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80 −/− mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80 −/− mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis.

MicroRNA (miRNA) deregulation plays a critical role in tumorigenesis. miR-23b and miR-130b are induced by thyrotropin in thyroid cells in a cAMP-dependent manner. The aim of our work has been to investigate the possible role of miR-23b and miR-130b in pituitary tumorigenesis. We have analyzed their expression in a panel of pituitary adenomas (PAs) including GH and NFPA adenomas. We report that miR-23b and miR-130b are drastically reduced in GH, gonadotroph and NFPA adenomas in comparison with normal pituitary gland. Interestingly, the overexpression of miR-23b and miR-130b inhibits cell proliferation arresting the cells in the G1 and G2 phase of the cell cycle, respectively. Moreover, we demonstrate that miR-23b and miR-130b target HMGA2 and cyclin A2 (CCNA2) genes, respectively. Finally, downregulation of miR-23b and miR-130b expression is associated with increased levels of their respective targets in human PAs. These findings suggest that miR-23b and miR-130b downregulation may contribute to pituitary tumorigenesis.

Context: Thyroid carcinoma is one of the most common malignancies of the endocrine system, and, despite the high frequency of oncogene activation in thyroid neoplastic lesions, the tumor suppressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicated a link between the CL2/CCDC80 gene and thyroid cancer. Objective: The objective of the study was to examine the expression of the CL2/CCDC80 gene in human thyroid carcinomas in the attempt to determine whether it plays a role in thyroid carcinogenesis. Design: We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissue samples differing in degree of malignancy. We also investigated the effects of its restoration in 2 human thyroid carcinoma cell lines characterized by very low levels of CL2/CCDC80 expression. Results: CL2/CCDC80 expression was much lower in almost all the thyroid carcinomas analyzed than in normal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozygosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restoration of CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a higher susceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression positively regulated the expression of E-cadherin, thereby halting cancer progression. Conclusions: These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroid carcinogenesis.

The overexpression of the HMGA1 proteins is a feature of human malignant neoplasias and has a causal role in cell transformation. The aim of our study has been to investigate the microRNAs (miRNAs or miRs) regulated by the HMGA1 proteins in the process of cell transformation analyzing the miRNA expression profile of v‐ras‐Ki oncogene‐transformed thyroid cells expressing or not HMGA1 proteins. We demonstrate that, among the miRNAs regulated by cell transformation, there are miR‐10b, miR‐21, miR‐125b, miR‐221 and miR‐222 that are positively and miR‐34a and miR‐603 that are negatively regulated by HMGA1 expression. Then, we focused our attention on the miR‐10b and miR‐603 whose expression was dependent on the presence of HMGA1 also in other cell systems. We found that miR‐10b is able to target the PTEN gene, whereas miR‐603 targets the CCND1 and CCND2 genes coding for the cyclin D1 and cyclin D2 proteins, respectively. Moreover, functional studies showed that miR‐10b and miR‐603 regulate positively and negatively, respectively, cell proliferation and migration suggesting a role of their dysregulation in thyroid cell transformation.

CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424) which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation. © 2010 Macmillan Publishers Limited All rights reserved.

Using gene expression profiling, we found that the CBX7 gene was drastically down-regulated in six thyroid carcinoma cell lines versus control cells. The aims of this study were to determine whether CBX7 is related to the thyroid cancer phenotype and to try to identify new tools for the diagnosis and prognosis of thyroid cancer. We thus evaluated CBX7 expression in various snap-frozen and paraffin-embedded thyroid carcinoma tissues of different degrees of malignancy by quantitative reverse transcription-PCR and immunohistochemistry, respectively. CBX7 expression progressively decreased with malignancy grade and neoplasia stage. Indeed, it decreased in an increasing percentage of cases going from benign adenomas to papillary (PTC), follicular, and anaplastic (ATC) thyroid carcinomas. This finding coincides with results obtained in rat and mouse models of thyroid carcinogenesis. CBX7 loss of heterozygosity occurred in 36.8% of PTC and in 68.7% of ATC. Restoration of CBX7 expression in thyroid cancer cells reduced growth rate, with a retention in the G1 phase of the cell cycle, suggesting that CBX7 can contribute to the proliferation of the transformed thyroid cells. In conclusion, loss of CBX7 expression correlates with a highly malignant phenotype in thyroid cancer patients. [Cancer Res 2008;68(16):6770–8]

We have recently reported that MicroRNAs (miR)-221 and miR-222 were up-regulated in human thyroid papillary carcinomas in comparison with the normal thyroid tissue. Bioinformatic analysis proposed the p27Kip1 protein, a key regulator of cell cycle, as a candidate target for the miR-221/222 cluster. Here, we report that the enforced expression of miR-221 and miR-222 was able to reduce p27Kip1 protein levels in thyroid carcinoma and HeLa cells in the absence of significant changes in specific p27Kip1 mRNA levels. This effect is direct as miR-221 and miR-222 negatively regulate the expression of the 3′-untranslated region-based reporter construct from the p27Kip1 gene, and is dependent on two target sites in this region. Consistent with these results, an enforced expression of the miR-221 and miR-222 induced the thyroid papillary carcinoma cell line (TPC-1) to progress to the S phase of the cell cycle. It is likely that the negative regulation of p27Kip1 by miR-221 and miR-222 might also have a role in vivo since we report an inverse correlation between miR-221 and miR-222 up-regulation and down-regulation of the p27Kip1 protein levels in human thyroid papillary carcinomas. Therefore, the data reported here demonstrate that miR-221 and miR-222 are endogenous regulators of p27Kip1 protein expression, and thereby, the cell cycle.

Thyroid carcinomas comprise a broad spectrum of tumors with different clinical behaviors. On the one side, there are occult papillary carcinomas (PTC), slow growing and clinically silent, and on the other side, rapidly growing anaplastic carcinomas (ATC), which are among the most lethal human neoplasms. We have analysed the microRNA (miR) profile of ATC in comparison to the normal thyroid using a microarray (miRNACHIP microarray). By this approach, we found an aberrant miR expression profile that clearly differentiates ATC from normal thyroid tissues and from PTC analysed in previous studies. In particular, a significant decrease in miR-30d, miR-125b, miR-26a and miR-30a-5p was detected in ATC in comparison to normal thyroid tissue. These results were further confirmed by northern blots, quantitative reverse transcription-PCR analyses and in situ hybridization. The overexpression of these four miRs in two human ATC-derived cell lines suggests a critical role of miR-125b and miR-26a downregulation in thyroid carcinogenesis, since a cell growth inhibition was achieved. Conversely, no effect on cell growth was observed after the overexpression of miR-30d and miR-30a-5p in the same cells. In conclusion, these data indicate a miR signature associated with ATC and suggest the miR deregulation as an important event in thyroid cell transformation. © 2007 Nature Publishing Group All rights reserved.