Unraveling the molecular architecture of autoimmune thyroid diseases at spatial resolution.

Autoimmune thyroid diseases (AITD) such as Graves' disease (GD) or Hashimoto's thyroiditis (HT) are organ-specific diseases that involve complex interactions between distinct components of thyroid tissue. Here, we use spatial transcriptomics to explore the molecular architecture, heterogeneity and location of different cells present in the thyroid tissue, including thyroid follicular cells (TFCs), stromal cells such as fibroblasts, endothelial cells, and thyroid infiltrating lymphocytes. We identify damaged antigen-presenting TFCs with upregulated CD74 and MIF expression in thyroid samples from AITD patients. Furthermore, we discern two main fibroblast subpopulations in the connective tissue including ADIRF+ myofibroblasts, mainly enriched in GD, and inflammatory fibroblasts, enriched in HT patients. We also demonstrate an increase of fenestrated PLVAP+ vessels in AITD, especially in GD. Our data unveil stromal and thyroid epithelial cell subpopulations that could play a role in the pathogenesis of AITD.

Analysis of T follicular and T peripheral helper lymphocytes in autoimmune thyroid disease.

PURPOSE: Peripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD. METHODS: We performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto's thyroiditis (HT), twenty-four with Graves' disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses. RESULTS: Increased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD. CONCLUSION: Our data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD.

Primary Cilia as a Tumor Marker in Pituitary Neuroendocrine Tumors.

Pituitary neuroendocrine tumors (PitNETs) account for approximately 15% of all intracranial neoplasms. Although they usually appear to be benign, some tumors display worse behavior, displaying rapid growth, invasion, refractoriness to treatment, and recurrence. Increasing evidence supports the role of primary cilia (PC) in regulating cancer development. Here, we showed that PC are significantly increased in PitNETs and are associated with increased tumor invasion and recurrence. Serial electron micrographs of PITNETs demonstrated different ciliation phenotypes (dot-like versus normal-like cilia) that represented PC at different stages of ciliogenesis. Molecular findings demonstrated that 123 ciliary-associated genes (eg, doublecortin domain containing protein 2, Sintaxin-3, and centriolar coiled-coil protein 110) were dysregulated in PitNETs, representing the upregulation of markers at different stages of intracellular ciliogenesis. Our results demonstrate, for the first time, that ciliogenesis is increased in PitNETs, suggesting that this process might be used as a potential target for therapy in the future.

Evaluation of Epithelial-Mesenchymal Transition Markers in Autoimmune Thyroid Diseases.

A state of chronic inflammation is common in organs affected by autoimmune disorders, such as autoimmune thyroid diseases (AITD). Epithelial cells, such as thyroid follicular cells (TFCs), can experience a total or partial transition to a mesenchymal phenotype under these conditions. One of the major cytokines involved in this phenomenon is transforming growth factor beta (TGF-ß), which, at the initial stages of autoimmune disorders, plays an immunosuppressive role. However, at chronic stages, TGF- ß contributes to fibrosis and/or transition to mesenchymal phenotypes. The importance of primary cilia (PC) has grown in recent decades as they have been shown to play a key role in cell signaling and maintaining cell structure and function as mechanoreceptors. Deficiencies of PC can trigger epithelial-mesenchymal transition (EMT) and exacerbate autoimmune diseases. A set of EMT markers (E-cadherin, vimentin, α-SMA, and fibronectin) were evaluated in thyroid tissues from AITD patients and controls through RT-qPCR, immunohistochemistry (IHC), and western blot (WB). We established an in vitro TGF-ß-stimulation assay in a human thyroid cell line to assess EMT and PC disruption. EMT markers were evaluated in this model using RT-qPCR and WB, and PC was evaluated with a time-course immunofluorescence assay. We found an increased expression of the mesenchymal markers α-SMA and fibronectin in TFCs in the thyroid glands of AITD patients. Furthermore, E-cadherin expression was maintained in these patients compared to the controls. The TGF-ß-stimulation assay showed an increase in EMT markers, including vimentin, α-SMA, and fibronectin in thyroid cells, as well as a disruption of PC. The TFCs from the AITD patients experienced a partial transition to a mesenchymal phenotype, preserving epithelial characteristics associated with a disruption in PC, which might contribute to AITD pathogenesis.

Impact of Change in Body Composition during Follow-Up on the Survival of GEP-NET.

Background: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous rare diseases causing malnutrition and cachexia in which the study of body composition may have an impact in prognosis. Aim: Evaluation of muscle and fat tissues by computed tomography (CT) at the level of the third lumbar (L3 level) at diagnosis and at the end of follow-up in GET-NET patients and their relationships with clinical and biochemical variables as predictors of survival. Methodology: Ninety-eight GEP-NET patients were included. Clinical and biochemical parameters were evaluated. Total body, subcutaneous, visceral and total fat areas and very low-density, low-density, normal density, high-density, very high-density and total muscle areas were obtained from CT images. Results: Body composition measures and overall mortality correlated with age, ECOG (Eastern Cooperative Oncology Group performance status) metastases, lactate dehydrogenase (LDH), albumin and urea levels. Although there was no relationship between body composition variables at diagnosis and overall and specific mortality, an increase in low-density muscle and a decrease in normal-density muscle during follow-up were independently correlated to overall (p <0.05) and tumor-cause mortality (p < 0.05). Conclusion: Although body composition measures obtained by CT at diagnosis did not impact survival of GEP-NET patients, a loss of good quality muscle during follow-up was associated with an increased overall and tumor-related mortality. Nutritional status should therefore be supervised by nutrition specialists and an increase in good quality muscle could improve prognosis.

Analysis of Expression of Different Histone Deacetylases in Autoimmune Thyroid Disease.

CONTEXT: Histone deacetylases (HDACs) and histone acetyltransferases (HAT) have an important role in the regulation of gene transcription as well as in the development and function of CD4+Foxp3+ T regulatory (Treg) cells. Our group and others have reported that patients with autoimmune thyroid disease (AITD) show abnormalities in the levels and function of different Treg cell subsets. OBJECTIVE: We aimed to analyze the levels of expression of several HDACs and the Tip60 HAT in the thyroid gland and immune cells from patients with AITD. METHODS: The expression of HDAC1-11 and the Tip60 HAT, at RNA and protein levels, were determined in thyroid tissue from 20 patients with AITD and 10 healthy controls and these findings were correlated with clinical data. HDAC9 and Tip60 levels were also analyzed in thyroid cell cultures, stimulated or not with proinflammatory cytokines, as well as in different cell subsets from peripheral blood mononuclear cells. RESULTS: Altered expression of different HDACs was observed in thyroid tissue from AITD patients, including a significant increase in HDAC9, at RNA and protein levels. Likewise, HDAC9 expression was increased in peripheral blood mononuclear cells particularly in Treg cells in patients with AITD. In contrast, Tip60 expression was reduced in thyroid gland samples from patients with Hashimoto thyroiditis. CONCLUSION: Our results indicate that HDAC expression is dysregulated in thyroid gland and immune cells from patients with AITD, suggesting involvement in the pathogenesis of this condition.

LAT-1 and GLUT-1 Carrier Expression and Its Prognostic Value in Gastroenteropancreatic Neuroendocrine Tumors.

Cancer cells develop mechanisms that increase nutrient uptake, including key nutrient carriers, such as amino acid transporter 1 (LAT-1) and glucose transporter 1 (GLUT-1), regulated by the oxygen-sensing Von Hippel Lindau-hypoxia-inducible factor (VHL-HIF) transcriptional pathway. We aimed to analyze these metabolic players in gastroenteropancreatic neuroendocrine tumors (GEP-NET) and correlate them with tumor malignancy and progression. LAT-1, GLUT-1, and pVHL expression was analyzed in 116 GEP-NETs and 48 peritumoral tissue samples by immunohistochemistry. LAT-1 was stably silenced using specific shRNA in the human NET BON cell line. LAT-1 expression was significantly increased in tumor tissue compared to non-tumor tissue in both gastrointestinal (67% vs. 44%) and pancreatic NETs (54% vs. 31%). Similarly, GLUT-1 was substantially elevated in gastrointestinal (74% vs. 19%) and pancreatic (58% vs. 4%) NETs. In contrast, pVHL expression was decreased (85% vs. 58%) in pancreatic NETs. Tumors with metastases at diagnosis displayed increased LAT-1 and GLUT-1 and decreased pVHL expression (p < 0.001). In accordance with these data, silencing LAT-1 curtailed cell proliferation in BON cells. These findings suggest that specific mechanisms that increase nutrient uptake, such as LAT-1 and GLUT-1, are increased in GEP-NETs, whereas pVHL is decreased. These markers might be related to the proliferation and metastatic capacity of these tumors.

Quantitative and Functional Analysis of PD-1+ NK Cells in Patients With Autoimmune Thyroid Disease.

CONTEXT: Natural killer (NK) cells have an important role in innate immunity and in the regulation of immune response. The role of NK cells expressing the programmed cell death protein-1 (PD-1) regulatory receptor has not been explored in patients with autoimmune thyroid disease (AITD). PURPOSE: To analyze the levels and function of PD-1+ NK cells in samples from AITD patients. DESIGN: Cases and controls, observational study. SETTING: Hospital Universitario la Princesa, Spain. PATIENTS: Forty patients with AITD, 16 with Hashimoto thyroiditis (HT), 24 with Graves' disease (GD), and 15 healthy controls. INTERVENTION: Multiparametric flow cytometry analysis of peripheral blood NK cells. In vitro assays of cytotoxic activity of NK cells, and synthesis of cytokines. MAIN OUTCOME MEASURES: Levels and function of PD-1+ NK cells in blood samples from AITD patients and controls. RESULTS: Increased levels of NK cells and the CD56dimPD-1+ subset were observed in GD patients. In HT, an enhanced expression of the regulatory receptors NKG2A and NKG2C by CD56brightPD-1+ NK cells was detected. AITD patients showed an increased synthesis of IL-10 by CD56brightPD-1- NK cells, whereas CD56dimPD-1+ cells from GD patients exhibited an enhanced production of interferon-γ. PD-1+ NK cells from patients with GD and HT showed an increased cytotoxic activity. Significant associations were observed in patients with GD or HT between the levels of PD-1+ NK cells and clinical laboratory parameters. CONCLUSIONS: The different abnormalities in NK cell subset levels, in the expression of PD-1 and its function in AITD patients' further support the complex role of these cells in this pathogenesis.

Utility of circulating serum miRNA profiles to evaluate the potential risk and severity of immune-mediated inflammatory disorders.

Immune-mediated inflammatory disorders (IMID) are a group of diseases that present inflammation as a major pathogenic mechanism. They affect 15% of the population and pose a heavy socio-economic burden. Despite the growing knowledge on the etiopathogenesis of these diseases and the marked improvement in their management, there is a lack of predictive markers of IMID development or severity suitable for early diagnosis and adjustment of treatment intensity. The possibility that certain circulating miRNA profiles could be used as biomarkers of risk of development and/or severity of several autoimmune diseases has fuelled the interest in using them to improve the selection of successful treatments. The multi-pronged approach proposed here sought to reveal circulating miRNAs and miRNA signatures that could act as new predictive biomarkers of IMID development and severity. Our results showed that the circulating levels of miR-19b and miR-26b were significantly decreased (p < 0.001) in IMID patients compared to controls. Furthermore, receiver operating characteristic (ROC) curve analysis showed that these miRNAs were suitable discriminators capable to identify an IMID, with areas under the curve (AUC) of 0.85 and 0.83, respectively. In addition, we established that miR-19a and miR-143 were significantly increased in IMID patients with severe disease (p < 0.05). In summary, our findings identify two different miRNA signatures. One of them is associated with the presence of IMIDs and could lead to the development of tools for their early detection. The second signature is able to discriminate between mild and severe forms of these disorders and could be a putative tool to select patient candidates for a more intense treatment.

Integrated miRNA and mRNA expression profiling identifies novel targets and pathological mechanisms in autoimmune thyroid diseases.

BACKGROUND: The mechanisms underlying autoimmune thyroid disease (AITD) remain elusive. Identification of such mechanisms would reveal novel and/or better therapeutic targets. Here, we use integrated analysis of miRNAs and mRNAs expression profiling to identify potential therapeutic targets involved in the mechanisms underlying AITD. METHODS: miRNA and mRNA from twenty fresh-frozen thyroid tissues (15 from AITD patients and 5 from healthy controls) were subjected to next-generation sequencing. An anti-correlated method revealed potential pathways and disease targets, including proteins involved in the formation of primary cilia. Thus, we examined the distribution and length of primary cilia in thyroid tissues from AITD and controls using immunofluorescence and scanning electron microscopy, and parsed cilia formation in thyroid cell lines in response to inflammatory stimuli in the presence of miRNA mimics. FINDINGS: We found that the expression of miR-21-5p, miR-146b-3p, miR-5571-3p and miR-6503-3p was anti-correlated with Enolase 4 (ENO4), in-turned planar cell polarity protein (INTU), kinesin family member 27 (KIF27), parkin co-regulated (PACRG) and serine/threonine kinase 36 (STK36) genes. Functional classification of these miRNA/mRNAs revealed that their differential expression was associated with cilia organization. We demonstrated that the number and length of primary cilia in thyroid tissues was significantly lower in AITD than in control (frequency of follicular ciliated cells in controls = 67.54% vs a mean of 22.74% and 21.61% in HT and GD respectively p = 0.0001, by one-way ANOVA test). In addition, pro-inflammatory cytokines (IFNγ and TNFα) and specific miRNA mimics for the newly identified target genes affected cilia appearance in thyroid cell lines. INTERPRETATION: Integrated miRNA/gene expression analysis has identified abnormal ciliogenesis as a novel susceptibility pathway that is involved in the pathogenesis of AITD. These results reflect that ciliogenesis plays a relevant role in AITD, and opens research pathways to design therapeutic targets in AITD. FUNDING: Instituto de Salud Carlos III, Comunidad de Madrid, Grupo Español de Tumores Neuroendocrinos y Endocrinos, Ministerio de Economía y Empresa and FEDER.

Analysis of expression of the PD-1/PD-L1 immune checkpoint system and its prognostic impact in gastroenteropancreatic neuroendocrine tumors.

The immune checkpoint based therapy targeting the programmed death-1 (PD-1) receptor and its PD-L1 ligand has recently been approved for the therapy of different malignant conditions, but not yet for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). In this context, we evaluated the expression of PD-1 and PD-L1 in GEP-NETs and its potential correlations with clinical outcomes. Expression of PD-1/PD-L1 was analyzed by immunohistochemistry in 116 GEP-NETs and 48 samples of peritumoral tissue. In addition, the expression of these molecules was assessed by flow cytometry in peripheral blood mononuclear cells (PBMC) from patients with GEP-NETs (n = 32) and healthy controls (n = 32) and in intratumoral mononuclear cells (TMCs) (n = 3). Expression of PD-L1 and PD-1 was detected by immunohistochemistry in 6% and 1% of tumor tissue samples, respectively, and in 8% of peritumoral tissue samples, for both markers. We also observed that PD-1 expression by TMCs was associated with metastatic disease at diagnosis, and the levels of circulating PD-1+ PBMCs were associated with progressive disease upon follow-ups. In addition, circulating PD-1+ PBMCs were significantly correlated with PD-L1 expression by tumor cells. Our data suggest that PD-1/PD-L1 is expressed in 1 to 8% of GEP-NETs, and that this feature is significantly associated with disease evolution (p < 0.01).

Patients With Autoimmune Thyroiditis Show Diminished Levels and Defective Suppressive Function of Tr1 Regulatory Lymphocytes.

Context: T regulatory type 1 (Tr1) cells are a subpopulation of T lymphocytes (CD4+CD49+LAG-3+IL-10+) that exert a considerable immunosuppressive effect. However, their possible role in autoimmune thyroid disease (AITD) has not been explored so far. Purpose: To analyze the levels and function of Tr1 cells in peripheral blood and thyroid tissue of patients with AITD. Design: Cases and controls, observational study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid, Spain. Patients: Thirty-eight patients with AITD (23 with Graves disease and 15 with Hashimoto thyroiditis) and 26 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques were used to analyze the levels in peripheral blood (n = 38) and thyroid mononuclear cells (n = 5). An in vitro assay of suppression of cellular activation and cytokine release was performed to study the function of Tr1 cells. Main Outcome Measure: Levels and function of Tr1 cells in patients with AITD and controls. Results: Levels of Tr1 cells were significantly diminished in peripheral blood from patients with AITD. Functional studies showed that Tr1 cells from patients with AITD exhibit a diminished suppressive function compared with healthy controls. Tr1 levels were associated with disease severity, including longer duration of the disease and ophthalmopathy activity, and with autoantibody titers. Conclusions: The low levels of Tr1 cells and their diminished function may have a relevant role in the defective immune-regulatory function characteristic of patients with AITD.

Hypophysitis following Treatment with Ustekinumab: Radiological and Pathological Findings.

CONTEXT: Ustekinumab is a human IgG1 monoclonal antibody that targets interleukin (IL)-12 and IL-23, which may be useful in the treatment of autoimmune conditions such as psoriasis, psoriatic arthritis, and Crohn's disease. Hypophysitis is an immune-derived inflammatory condition of the pituitary gland that may lead to pituitary dysfunction. With the increasing use of immunotherapy, it is possible that this and other new immune-related adverse events (IRAEs) arise, although the mechanisms involved are still incompletely defined. CASE DESCRIPTION: A 35-year-old male, with a previous history of severe plaque-psoriasis who had started treatment with ustekinumab 4 months before, complained of progressive and persistent headache. Brain magnetic resonance imaging (MRI) was unremarkable. One year later, a new MRI was performed due to headache persistence, which revealed a homogenous and diffuse pituitary enlargement, with suprasellar extension and optic chiasm involvement, blurring of the pituitary stalk, absence of clear differentiation between the anterior and posterior lobes, and no signs of hemorrhage or adenomas. Endocrine evaluation was consistent with panhypopituitarism. Work-up of infiltrative and infectious diseases was negative. Follow-up MRI revealed an increase in the pituitary enlargement and transsphenoidal surgery was performed. Pathological findings revealed an intense fibrosis and a chronic inflammatory infiltrate, but no evidence of adenoma, granuloma, or acid fast bacilli. Immunohistochemical staining showed a combined T-cell (CD3+, CD4+) and B-cell (CD19+, CD20+) phenotype. CONCLUSION: We suggest a novel IRAE of ustekinumab, with full radiological and immunopathological iconography, which may be mediated by the complex interaction between different immunological processes.

A MicroRNA Signature for Evaluation of Risk and Severity of Autoimmune Thyroid Diseases.

Context: Circulating microRNAs (miRNAs) are emerging as an interesting research area because of their potential role as novel biomarkers and therapeutic targets. Their involvement in autoimmune thyroid diseases (AITDs) has not been fully explored. Objective: To compare the expression profile of miRNAs in thyroid tissue from patients with AITD and controls, using next-generation sequencing, further validated our findings in thyroid and serum samples. Design: Twenty fresh-frozen thyroid tissues (15 from patients with AITD and 5 from controls) were used for miRNA next-generation sequencing. Thirty-six thyroid samples were recruited for the qRT-PCR validation test and 58 serum samples for further validation in peripheral blood. Results: Expression of several miRNAs that had been previously associated with relevant immunological functions was significantly dysregulated. Specifically, eight differentially expressed miRNAs (miR-21-5p, miR-142-3p, miR-146a-5p, miR-146b-5p, miR-155-5p, miR-338-5p, miR-342-5p, and miR-766-3p) were confirmed using qRT-PCR in thyroid samples, and three had the same behavior in tissue and serum samples (miR-21-5p, miR-142-3p, and miR-146a-5p). Furthermore, when the expression of these miRNAs was assessed together with five additional ones previously related to AITD in peripheral blood, the expression of five (miR-Let7d-5p, miR-21-5p, miR-96-5p, miR-142-3p, and miR-301a-3p) was significantly expressed in AITD and, in patients with Graves disease (GD), was correlated with a higher severity of disease, including active ophthalmopathy, goiter, higher antibody titers, and/or higher recurrence rates. Conclusions: The present findings identify a serum five-signature miRNA that could be an independent risk factor for developing AITD and a predisposition of a worse clinical picture in patients with GD.

Pathogenic Th17 and Th22 cells are increased in patients with autoimmune thyroid disorders.

PURPOSE: To study the levels of pathogenic and non-pathogenic Th17 and Th22 cells in autoimmune thyroid disorders patients. Although Th17 cells seem to play an important role in the pathogenesis of thyroid autoimmune disorders, the specific subsets of these lymphocytes have not been analyzed in this condition. METHODS: We assessed the levels of Th17 (pathogenic and non-pathogenic) and Th22 cells in peripheral blood and thyroid glands of autoimmune thyroid disorders patients (n = 26, 16 with Graves' disease and 10 with Hashimoto's thyroiditis) and 15 healthy controls by multi-parametric flow cytometry and immunofluorescence microscopy. RESULTS: We found increased levels of pathogenic Th17 lymphocytes and Th22 cells in peripheral blood from autoimmune thyroid disorders patients. In addition, these cells were detected in thyroid glands from HT patients. Furthermore, we found significant correlations between the levels of these cells and disease activity, disease duration, and the presence of ophthalmopathy. CONCLUSIONS: The increased levels of pathogenic Th17 lymphocytes and Th22 cells in autoimmune thyroid disorders suggest their involvement in the pathogenesis of this condition.

Expression and Function of the Costimulatory Receptor SLAMF1 Is Altered in Lymphocytes From Patients With Autoimmune Thyroiditis.

Context: Signaling lymphocytic activation molecule family 1 (SLAMF1) is a costimulatory receptor expressed by most immune cells. Its role in autoimmune thyroid disease (AITD) is not well known. Objective: To analyze the expression and function of the costimulatory receptor SLAMF1 in lymphocytes of patients with AITD. Design: Cross-sectional, prospective, single-center study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid. Patients: Twenty-eight patients with AITD (17 with Graves disease and 11 with Hashimoto thyroiditis) and 21 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques to analyze the expression of SLAMF1 in peripheral blood (n = 28) and thyroid tissue (n = 5) mononuclear cells. Assay of inhibition of cellular proliferation to study the function of SLAMF1 in CD4+CD25+ T regulatory (Treg) cells. Main Outcome Measure: Expression levels and the function of SLAMF1 in lymphocytes in AITD patients and controls. Results: Expression of SLAMF1 was significantly increased in peripheral blood CD4+, T helper 17, and CD19+ B cells from AITD patients. Immunofluorescence microscopy detected the presence of SLAMF1+ lymphocytes in thyroid inflammatory cell infiltrate. Functional studies showed that SLAMF1 engagement in Treg cells increased their suppressive function in healthy controls but not in AITD patients. Conclusions: The altered expression of SLAMF1, as well as its defective function observed in patients with AITD, may have a relevant role in the defective immune-regulatory function observed in this condition.

Presence of sst5TMD4, a truncated splice variant of the somatostatin receptor subtype 5, is associated to features of increased aggressiveness in pancreatic neuroendocrine tumors.

PURPOSE: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous tumors, and their biological behavior is not well known. We studied the presence and potential functional roles of somatostatin receptors (sst1-5), focusing particularly on the truncated variants (sst5TMD4, sst5TMD5) and on their relationships with the angiogenic system (Ang/Tie-2 and VEGF) in human GEP-NETs. EXPERIMENTAL DESIGN: We evaluated 42 tumor tissue samples (26 primary/16 metastatic) from 26 patients with GEP-NETs, and 30 non-tumoral tissues (26 from adjacent non-tumor regions and 4 from normal controls) from a single center. Expression of sst1-5, sst5TMD4, sst5TMD5, Ang1-2, Tie-2 and VEGF was analyzed using real-time qPCR, immunofluorescence and immunohistochemistry. Expression levels were associated with tumor characteristics and clinical outcomes. Functional role of sst5TMD4 was analyzed in GEP-NET cell lines. RESULTS: sst1 exhibited the highest expression in GEP-NET, whilst sst2 was the most frequently observed sst-subtype (90.2%). Expression levels of sst1, sst2, sst3, sst5TMD4, and sst5TMD5 were significantly higher in tumor tissues compared to their adjacent non-tumoral tissue. Lymph-node metastases expressed higher levels of sst5TMD4 than in its corresponding primary tumor tissue. sst5TMD4 was also significantly higher in intestinal tumor tissues from patients with residual disease of intestinal origin compared to those with non-residual disease. Functional assays demonstrated that the presence of sst5TMD4 was associated to enhanced malignant features in GEP-NET cells. Angiogenic markers correlated positively with sst5TMD4, which was confirmed by immunohistochemical/fluorescence studies. CONCLUSIONS: sst5TMD4 is overexpressed in GEP-NETs and is associated to enhanced aggressiveness, suggesting its potential value as biomarker and target in GEP-NETs.

Levels of regulatory T cells CD69(+)NKG2D(+)IL-10(+) are increased in patients with autoimmune thyroid disorders.

Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4(+)CD69(+) and CD4(+)NKG2D(+) T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4(+)CD69(+)IL-10(+), CD4(+)CD69(+)NKG2D(+), and CD4(+)CD69(+)IL-10(+)NKG2D(+) cells, in peripheral blood from GD patients compared to controls. The increase in CD4(+)CD69(+)IL-10(+) and CD4(+)CD69(+)IL-10(+)NKG2D(+) T cells was especially remarkable in patients with active Graves' ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4(+)CD69(+)IL-10(+) or CD4(+)CD69(+)IL-10(+)NKG2D(+) cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69(+) regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69(+), NKG2D(+), and IL-10(+) cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69(+) Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69(+) regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.

Circulating Microvesicles Regulate Treg and Th17 Differentiation in Human Autoimmune Thyroid Disorders.

BACKGROUND: Microvesicles (MVs) are emerging as important contributors to the development of inflammatory and autoimmune diseases. MVs can mediate immune modulation carrying genetic information, including microRNAs that can be transferred between cells. DESIGN: We determined the plasma levels of annexin-V+ MVs derived from different immune cells and platelets in patients with autoimmune thyroid diseases (AITDs) and in healthy controls. T lymphocyte polarization assays were performed in the presence of MVs to evaluate their effect in T regulatory and T helper 17 cells differentiation. microRNA content into plasma MVs and their corresponding mRNA targets were evaluated by RT-PCR. RESULTS: The percentage of platelet-derived MVs (CD41a+) was significantly increased in plasma samples from AITD patients compared with healthy controls. In contrast, patients with AITD showed a lower percentage of leukocyte and endothelial cell-derived MVs compared with controls. In addition, functional assays showed that MVs from AITD patients inhibited the in vitro differentiation of Foxp3+ T regulatory cells (11.35% vs 4.40%, P = .01) and induced the expression of interferon-γ by CD4+ lymphocytes (10.91% vs 13.99%, P = .01) as well as the differentiation of T helper 17 pathogenic (IL-17+interferon-γ+) cells (1.98% vs 5.13%, P = .03). Furthermore, in AITD patients, whereas miR-146a and miR-155 were increased in circulating MVs, their targets IL-8 and SMAD4 were decreased in peripheral blood mononuclear cells. CONCLUSIONS: Our data indicate that circulating MVs seem to have a relevant role in the modulation of the inflammatory response observed in AITD.