Pierina Richiusa

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 (64±9% and 57±12%, 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.

BRAFV600E mutation and p27kip1 expression in papillary carcinomas of the thyroid ≤1 cm and their paired lymph node metastases

BRAFV600E mutation and p27kip1 expression have been introduced as novel indicators that may predict prognosis in different tumors, as well as in papillary thyroid carcinomas.

Defective expression of the apoptosis-inducing CD95 (Fas/APO-1) molecule on T and B cells in IDDM

SummaryTriggering of CD95 (Fas/APO-1) cell surface receptors regulates the elimination of autoreactive T and B lymphocytes through a mechanism of cell suicide called apoptosis. Three different mutations involving CD95 or its ligand are responsible for induction of autoimmunity in susceptible mouse strains. To determine whether a defect involving the CD95 receptor is associated with human insulin-dependent diabetes mellitus (IDDM), we have studied the expression of CD95 on peripheral blood mononuclear cells from IDDM patients at different stages of the disease. Three-colour flow cytometry and mean fluorescence analysis showed that T and B lymphocytes from newly diagnosed IDDM and patients with long-standing disease, and subjects at high risk of developing the disease were highly defective in CD95 expression (p<0.001), whereas monocytes from all the groups studied expressed normal amounts of CD95 molecules on their cell surface. T-cell subset analysis showed that the impairment of CD95 expression in IDDM patients and high-risk subjects involved both CD3+ CD4+ (p<0.001) and CD3+ CD8+ cells (p range: <0.01–0.001), suggesting that this alteration concerns both helper and cytotoxic T cells. Moreover, after activation in vitro with anti-CD3 monoclonal antibody, T cells from newly diagnosed IDDM patients maintained a reduced CD95 expression during the entire cell culture period (24–72 h) in comparison to the control population (p<0.001). In conclusion, we found a reduced expression of the apoptosis-inducing CD95 receptor on T and B lymphocytes of individuals with clinical and preclinical IDDM. We hypothesize that this defective expression may impair the capacity of autoreactive lymphocytes to undergo CD95-mediated apoptosis, contributing to the lack of control on beta-cell specific B- and T-cell clones.

Multiple pluripotent stem cell markers in human anaplastic thyroid cancer: the putative upstream role of SOX2.

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 (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off. METHODS In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using 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±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and 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 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p<0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p<0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, 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.

BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy

BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.

Descriptive Epidemiology of Human Thyroid Cancer: Experience From a Regional Registry and The “Volcanic Factor”

Thyroid cancer (TC), the most common endocrine tumor, has steadily increased worldwide due to the increase of the papillary histotype. The reasons for this spread have not been established. In addition to more sensitive thyroid nodule screening, the effect of environmental factors cannot be excluded. Because high incidences of TC were found in volcanic areas (Hawaii and Iceland), a volcanic environment may play a role in the pathogenesis of TC. In January 2002, the Regional Register for TC was instituted in Sicily. With a population of approximately five million inhabitants with similar genetic and lifestyle features, the coexistence in Sicily of rural, urban, industrial, moderate-to-low iodine intake, and volcanic areas provides a conducive setting for assessing the environmental influences on the etiology of TC. In Sicily, between 2002 and 2004, 1,950 new cases of TC were identified, with an age-standardized rate (world) ASR(w) = 17.8/105 in females and 3.7/105 in males and a high female/male ratio (4.3:1.0). The incidence of TC was heterogeneous within Sicily. There were 2.3 times more cases in the Catania province (where most of the inhabitants live in the volcanic area of Mt. Etna): ASR(w) = 31.7/105 in females and 6.4/105 in males vs. 14.1 in females and 3.0 in males in the rest of Sicily. Multivariate analysis documented that residents in the volcanic area of Mt. Etna had a higher risk of TC, compared to the residents in urban, industrial, and iodine deficient areas of Sicily. An abnormally high concentration of several chemicals was found in the drinking water of the Mt. Etna aquifer, which provides water to most of the residents in the Catania province. Our data suggest that environmental carcinogen(s) of volcanic origin may promote papillary TC. Additional analyses, including cancer biological and molecular features, will allow a better understanding of risk factors and etiopathogenetic mechanisms.

Study of T-cell activation in type I diabetic patients and pre-type I diabetic subjects by cytometric analysis: antigen expression defect in vitro.

In Type I diabetes the observation of a decreased release of interleukin-2 (IL-2) and soluble IL-2 receptors by means of stimulated lymphocytesin vitro indicates that a primary immunoregulatory defect may be involved. To confirm this hypothesis we investigated the T-cell activation trend, evaluating the surface expression of IL-2 receptor (CD25), transferrin (CD71), HLA class II (DR), and CD69 phenotypes afterin vitro stimulation with phytohemagglutinin (PHA; 1 and 10 µg/ml) and concanavalin A (12.5 µg/ml) in six newly diagnosed Type I diabetics and six islet cell- and insulin autoantibody-positive first-degree relatives. As controls were studied six long-standing Type I diabetics and six healthy subjects. T-cell cultures from the four groups were performed on the same day and examined at 0, 24, 48, 96, 120, and 144 hr. Cytometric analysis was performed, keeping PBMC gating constant on the basis of physical parameters (scatter and volume). Using both PHA concentrations, a lower level of CD25, CD71, CD69, and DR antigen expression was found in newly diagnosed patients at all observation times with respect to control cultures (P<0.001). Unexpectedly, pre-Type I diabetic subjects, after 1 µg/ml of PHA, showed a significantly reduced expression of CD69 (P<0.001) and CD71 (P<0.001). The levels remained low, also with high PHA, at the different observation periods, while CD25 expression was found to be reduced in prediabetics only after 1 µg/ml of PHA (P<0.001). The long-standing patients showed a T cell activation trend very close to the latter. Our data show that in Type I diabetes and in the early phases of the disease, the initial activation signal(s) appears to be affected, particularly with one or more subsequent events necessary to initiate the appearance of “activation antigens.” This study suggests that the natural history of immunoregulation in pre-Type I and Type I diabetes is characterized by a primary defect in this system, which also persists in patients with long-standing disease.

Multimodal Surgical and Medical Treatment for Extensive Rhinocerebral Mucormycosis in an Elderly Diabetic Patient: A Case Report and Literature Review

Diabetes is a well-known risk factor for invasive mucormycosis with rhinocerebral involvement. Acute necrosis of the maxilla is seldom seen and extensive facial bone involvement is rare in patients with rhino-orbital-cerebral mucormycosis. An aggressive surgical approach combined with antifungal therapy is usually necessary. In this report, we describe the successful, personalized medical and surgical management of extensive periorbital mucormycosis in an elderly diabetic, HIV-negative woman. Mono- or combination therapy with liposomal amphotericin B (L-AmB) and posaconazole (PSO) and withheld debridement is discussed. The role of aesthetic plastic surgery to preserve the patients physical appearance is also reported. Any diabetic patient with sinonasal disease, regardless of their degree of metabolic control, is a candidate for prompt evaluation to rule out mucormycosis. Therapeutic and surgical strategies and adjunctive treatments are essential for successful disease management. These interventions may include combination therapy. Finally, a judicious multimodal treatment approach can improve appearance and optimize outcome in elderly patients.

Low bcl-2 expression and increased spontaneous apoptosis in T-lymphocytes from newly-diagnosed IDDM patients

SummaryThe bcl-2 gene product has been shown to regulate apoptotic cell death, and its dysregulation has been shown to induce several abnormalities in the immune system. No data exist regarding bcl-2 expression in autoimmune diseases, such as human insulin-dependent diabetes mellitus (IDDM). We investigated bcl-2 protein expression by testing T lymphocytes from 15 newly-diagnosed (<3 weeks) IDDM patients in comparison to 10 age-matched control subjects. The expression of bcl-2 on CD3+ lymphocyte subsets was investigated after membrane permeabilization by two- or three-colour immunofluorescence. When the percentage and mean fluorescence intensity (MFI) of bcl-2+/CD3+ cells from normal individuals and patients were compared, we found that bcl-2 expression within the CD3+ and CD4+ CD45R0+ T-cell populations was reduced significantly in IDDM patients (46.8±15.4 vs 79.6±11.7; 25.7±3.8 vs 47.15±5.7, respectively; p<0.001). To establish whether low bcl-2 expression in T cells from newly-diagnosed patients reflects their susceptibility to death by an apoptotic process, we also evaluated DNA staining with propidium iodide in CD3+ lymphocyte suspension after a (24–72 h) culture period (spontaneous apoptosis). We found that IDDM patients have higher levels of spontaneous apoptosis (mean±SEM: 24 h=4.6±0.8; 48 h=9.9±1; 72 h=12.8±1.1) than control subjects (24 h=1.8±0.4; 48 h=4.6±0.4; 72 h=5.7±0.3; p<0.02–0.001). Our study suggests that recent onset IDDM is characterised by reduced bcl-2 expression, which in turn may be associated with the increased spontaneous apoptosis we observed.

Neck Lymph Nodes in Chronic Autoimmune Thyroiditis: The Sonographic Pattern

BACKGROUND Neck lymph nodes may be involved in the pathogenesis of chronic autoimmune thyroiditis (CAT). This study was undertaken to identify which of the sonographic features of cervical lymph nodes are readily applicable to patients affected by CAT compared to healthy control subjects. METHODS We recruited 106 patients (92 females and 14 males) with CAT and 70 control subjects (53 females and 17 males) without clinical, biochemical, and ultrasonographic evidence of thyroid and neck diseases. We performed laboratory tests (thyrotropin, antithyroperoxidase antibodies, antithyroglobulin antibodies, and ultrasonography) to evaluate in each group: (i) thyroid function, autoimmunity, and morphology; (ii) number, topographic distribution (levels I-VI), and morphology of neck nodes (long-axis diameter; short-axis diameter; short-axis/long-axis ratio; absence or presence of hilus). RESULTS Total number of neck nodes with long-axis diameter >10 mm was significantly higher in the CAT group than in the control group (mean±standard deviation [SD]: 3.7±2.4 vs. 0.8±1.3; p<0.001) with significantly increased differences in levels II (1.4±0.8 vs. 0.3±0.5; p<0.001), III (2±1.2 vs. 0.3±0.7; p<0.001), and IV (0.7±0.7 vs. 0.07±0.2; p<0.001). More nodes with a hilus were found in the CAT group than in the control group (mean number of nodes±SD: 2.8±1.9 vs. 0.7±1.1; p<0.001). Short-axis diameter of level III (4.4±1 vs. 3.7±1.2 mm; p=0.002) and level IV nodes (3.9±1 vs. 3.1±0.5 mm p=0.030) was increased in CAT patients when compared with healthy controls. CONCLUSIONS The present study is the first one aiming at a systematic description of the sonographic pattern of cervical lymph nodes in CAT. An increased number of benign hyperplastic neck nodes, especially in levels II-IV, appears to be a characteristic sonographic finding associated with CAT.