Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1ß (IL-1ß) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.

SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.

Control of Erwinia amylovora Growth by Moringa oleifera Leaf Extracts: In Vitro and in Planta Effects.

Erwinia amylovora (EA) is a phytopathogenic bacterium, the causative agent of bacterial fire blight, a disease that affects Rosaceaes. In order to replace antibiotics and copper, the antimicrobial activity of three extracts of Moringa oleifera Lam., methanolic (MeOH-MOE), hydroalcoholic (HA-MOE) and hydroalcoholic with maltodextrins (HAMD-MOE), was tested on eleven strains of EA isolated from apple trees by the Emilia-Romagna Phytosanitary Department. MIC and MBC have been evaluated; biofilm formation, swarming motility and amylovoran production were performed with the crystalviolet, soft-agar assay and the amylovoran method. All extracts demonstrated bacteriostatic activity at a concentration of 1 mg/mL, resulting in a 80% reduction in biofilm formation. HAMD-MOE, MeOH-MOE and HA-MOE caused an inhibition of motility of 60%, 65% and 30% after 6 days and a decrease in amylovoran synthesis of 84%, 63% and 93%, respectively. In planta results showed how the compounds were able to inhibit EA virulence on apple trees, mainly if they were applied as a preventive treatment, although the treatment showed a significant reduction in fire blight symptoms progression. The antibacterial activity of the extracts is mainly due to the high concentration of polyphenolic compounds detected in the extracts that was able to alter the permeability of bacterial membrane, resulting in slowing the synthesis of ATP and consequently of all ATP-dependent functions, such as motility and less selectivity towards harmful compounds, which can, thus, enter the cytoplasm and inhibit enzymes involved in replication and quorum sensing. The efficacy, eco-compatibility and low cost make such extracts a potential tool for the control of bacterial fire blight.

Effects of Moringa oleifera Leaf Extracts on Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium belonging to the Xanthomonodaceae family, causing black rot in crucifers. To control this pathogen, the study investigated the effect of different leaves extracts of Moringa oleifera Lam., a tropical plant, well known for its food properties and with countless applications in many different fields, from nutraceutical (hypoglycemic) to the cosmetic (sunscreen) properties. Nevertheless, several studies pointed to its antibacterial action against both Gram-negative and Gram-positive bacteria. Many bioactive compounds, including flavonoids, phenolic acids, alkaloids, isothiocyanates, tannins and saponins, contained in these extracts, are responsible for its countless activities. The analyses carried out in this study show that the methanolic, hydroalcoholic and hydroalcoholic maltodextrin extracts have both bacteriostatic and bactericidal effects at concentrations of 0.5, 0.5 and 0.1 mg/mL respectively. In particular, the study shows how all extracts can alter membrane permeability, to adversely affect swarming motility, and to alter biofilm formation in Xcc. The in planta experiments showed a reduction of the necrosis area in the infected radishes, although the ability of the extracts to be absorbed by root systems is yet to be understood, in order to reach the target point.

PI3K/Akt/mTOR pathway involvement in regulating growth hormone secretion in a rat pituitary adenoma cell line.

PURPOSE: Insulin-like growth factor 1 (IGF1) controls growth hormone (GH) secretion via a negative feed-back loop that may disclose novel mechanisms possibly useful to control GH hyper-secretion. Our aim was to understand whether PI3K/Akt/mTOR pathway is involved in IGF1 negative feedback on GH secretion. METHODS: Cell viability, GH secretion, Akt, and Erk 1/2 phosphorylation levels in the rat GH3 cell line were assessed under treatment with IGF1 and/or everolimus, an mTOR inhitior. RESULTS: We found that IGF1 improves rat GH3 somatotroph cell viability via the PI3K/Akt/mTOR pathway and confirmed that IGF1 exerts a negative feedback on GH secretion by a transcriptional mechanism. We demonstrated that the negative IGF1 loop on GH secretion requires Akt activation that seems to play a pivotal role in the control of GH secretion. Furthermore, Akt activation is independent of PI3K and probably mediated by mTORC2. In addition, we found that Erk 1/2 is not involved in GH3 cell viability regulation, but may have a role in controlling GH secretion, independently of IGF1. CONCLUSION: Our data confirm that mTOR inhibitors may be useful to reduce pituitary adenoma cell viability, while Erk 1/2 pathway may be considered as a useful therapeutic target to control GH secretion. Our results open the field for further studies searching for effective drugs to control GH hyper-secretion.

Proposal for a novel management of indeterminate thyroid nodules on the basis of cytopathological subclasses.

Indeterminate thyroid nodules include heterogeneous lesions that could benefit from a differential management. Our aim is to better define the management of the Bethesda System for Reporting Thyroid Cytopathology class III and IV nodules, by identifying cytological subcategories among Bethesda System for Reporting Thyroid Cytopathology class III associated with different clinical risk, by means of ultrasound, repeated FNAB, and BRAFV600E molecular analysis. We also evaluated the outcome of nodules not operated, over a 5-year follow-up. Out of 460 nodules (269 Bethesda System for Reporting Thyroid Cytopathology class III and 191 Bethesda System for Reporting Thyroid Cytopathology class IV), 344 were operated on surgical group and 116 followed-up conservatively (follow-up group). Bethesda System for Reporting Thyroid Cytopathology class III was divided into four subcategories on the basis of cytomorphological features (III-1, III-2, III-3, III-4). Clinical risk was defined on the basis of histological, cytological, and ultrasound data. Malignancy was higher in Bethesda System for Reporting Thyroid Cytopathology class III vs. Bethesda System for Reporting Thyroid Cytopathology class IV (34.4 vs. 26.2 %; p < 0.01). Papillary thyroid carcinoma was the most frequent cancer in each Bethesda System for Reporting Thyroid Cytopathology class (35 %). BRAFV600E diagnostic accuracy was 87 %. Repeated FNAB reclassified as benign nearly 40 % of nodules, selecting patients where surgery could be spared. Significant nodule growth occurred in 13.7 % of nodules, belonging mostly to Bethesda System for Reporting Thyroid Cytopathology class III-2 and Bethesda System for Reporting Thyroid Cytopathology class IV. Overall clinical risk was higher in Bethesda System for Reporting Thyroid Cytopathology III-1, III-4, and IV classes. We propose a differential management of Bethesda System for Reporting Thyroid Cytopathology III and IV classes and related subcategories: surgery may be indicated in Bethesda System for Reporting Thyroid Cytopathology class III-1, III-4, and IV; a conservative follow-up avoiding repeated FNAB may be appropriated in class III-3, while repeated FNAB may be useful in class III-2.

Early onset acromegaly associated with a novel deletion in CDKN1B 5'UTR region.

Genetic alterations frequently are involved in the development of a pituitary adenoma in young age. We here characterize the functional role of a deletion in CDKN1B 5'-UTR region (c.-29_-26delAGAG) identified in an acromegalic patient that developed a growth hormone in pituitary adenoma during childhood. Our results show that the identified novel heterozygous deletion in the CDKN1B 5'-UTR region associates with a reduction in CDKN1B mRNA levels, a predicted altered secondary mRNA structure, and a reduced CDKN1B 5'-UTR transcriptional activity in vitro. The patient displayed loss of heterozygosity in the same CDKN1B 5'-UTR region at tissue level and the 5'UTR region containing the deleted sequence encompasses a GRE. These findings indicate that the identification of functional alterations of newly discovered genetic derangements need to be fully characterized and always correlated with the clinical manifestations.

Relevance of BRAF(V600E) mutation testing versus RAS point mutations and RET/PTC rearrangements evaluation in the diagnosis of thyroid cancer.

BACKGROUND: A molecular profile including BRAF and RAS mutations as well as RET/PTC rearrangement evaluation has been proposed to provide an accurate presurgical assessment of thyroid nodules and to reduce the number of unnecessary diagnostic surgeries, sparing patients' health and saving healthcare resources. However, the application of such molecular analyses may provide different results among different centers and populations in real-life settings. Our aims were to evaluate the diagnostic utility of assessing the presence of BRAF and RAS mutations and RET/PTC1 and RET/PTC3 rearrangements in all cytological categories in an Italian group of thyroid nodule patients assessed prospectively, and to understand whether and which mutation testing might be helpful in cytologically indeterminate nodules. METHODS: A total of 911 patients were submitted to ultrasound and fine-needle aspiration biopsy examination. Cytological evaluation was performed in parallel with molecular testing and compared to pathological results in 940 thyroid nodules, including 140 indeterminate lesions. RESULTS: BRAF mutation testing provided the best contribution to cancer diagnosis, allowing the disease to be detected at an early stage, and identifying indeterminate nodules in which diagnostic lobectomy could be spared. On the contrary, RAS and RET/PTC analysis did not further increase diagnostic sensitivity for thyroid cancer. In addition, we found RET/PTC rearrangements in benign lesions, indicating that this molecular marker might not be useful for the detection of thyroid cancer. CONCLUSION: BRAF(V600E) mutation analysis is superior to RAS point mutations and evaluation of RET/PTC rearrangements in the diagnosis of thyroid cancer, even in indeterminate lesions.

Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion.

Dopamine (DA) therapy of non-functioning pituitary adenomas (NFA) can result in tumor stabilization and shrinkage. However, the mechanism of action is still unknown. Previous evidence showed that DA can inhibit pituitary vascular endothelial growth factor expression (VEGF), that may be involved in pituitary tumor growth. The aim of our study was to clarify whether VEGF secretion modulation might mediate the effects of DA agonists on cell proliferation in human NFA. We assessed DA receptor subtype 2 (DR2) expression in 20 NFA primary cultures, where we also investigated the effects of a selective DR2 agonist, cabergoline (Cab), on VEGF secretion and on cell viability. All NFA samples expressed α-subunit and DR2 was expressed in 11 samples. In DR2 expressing tumors, Cab significantly reduced cell viability (-25%; P < 0.05) and VEGF secretion (-20%; P < 0.05). These effects were counteracted by treatment with the DA antagonist sulpiride. Cab antiproliferative effects were blocked by VEGF. Our data demonstrate that Cab, via DR2, inhibits cell viability also by reducing VEGF secretion in a selected group of NFA, supporting that DA agonists can be useful in the medical therapy of DR2 expressing NFA.

Role of ultrasonographic/clinical profile, cytology, and BRAF V600E mutation evaluation in thyroid nodule screening for malignancy: a prospective study.

CONTEXT: Ultrasound (US)-guided fine-needle aspiration biopsy (FNAB) is the most reliable nonsurgical test for distinguishing benign from malignant thyroid nodules. However, there is no consensus on which nodules should undergo FNAB. AIMS: The aims of this study were to evaluate the utility of US-guided FNAB in the diagnostic assessment of nodules with or without clinical/US features suggestive for malignancy and to investigate the additional contribution of BRAF V600E mutation analysis in the detection of differentiated thyroid cancer. DESIGN AND METHODS: Thyroid cytoaspirates from 2421 nodules at least 4 mm in diameter were performed in 1856 patients who underwent cytological evaluation and biomolecular analysis. RESULTS: Cytology showed high positive predictive value and specificity for the diagnosis of malignant lesions. BRAF V600E mutation was found in 115 samples, 80 of which were also cytologically diagnosed as papillary thyroid cancer. BRAF mutation analysis significantly enhanced the diagnostic value of cytology, increasing FNAB diagnostic sensitivity for malignant nodules by approximately 28%. Micro PTC (63% of diagnosed papillary thyroid carcinoma) showed a high prevalence of multifocality, extrathyroidal extension, and lymph node metastases, underlining the malignant potential of thyroid microcarcinomas. Each investigated US/clinical characteristic of suspected malignancy correlated with the presence of a thyroid cancer in thyroid nodules with diameter of at least 4 mm. CONCLUSIONS: These data indicate that nodules of at least 4 mm may underlie a thyroid cancer independently of US/clinical characteristics of suspected malignancy, suggesting the need to perform FNAB. The diagnostic sensitivity for thyroid cancer is significantly increased by BRAF V600E mutation analysis, indicating that the screening for BRAF mutation in FNAB samples has a relevant diagnostic potential.

Role of pituitary tumour transforming gene 1 in medullary thyroid carcinoma.

BACKGROUND: pituitary tumour transforming gene 1 (PTTG1) is over-expressed in a variety of endocrine-related tumours. We aimed at evaluating PTTG1 expression and function in human neoplastic parafollicular C-cells, represented by medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH) samples and by the TT cell line. METHODS: TT cells and tissues derived from human CCH (8 samples) and MTC (12 samples) were analyzed by northern blot, furthermore TT cells were subjected to PTTG gene silencing and cells were analyzed for DNA synthesis. RESULTS: PTTG1 expression was significantly higher (p<0.01) in CCH (3-fold), in papillary thyroid cancer and in MTC (5-fold) than in normal thyroid, and in MTC lymph-node metastases as compared to primary lesions (approximately 2-fold; p<0.05). PTTG1 mRNA and protein correlated with tumour diameter and TNM status (p<0.05). In TT cells, PTTG1 silencing did not completely block DNA synthesis, but significantly reduced [3H]Thymidine incorporation (~50%; p<0.01) for up to 3 days. CONCLUSIONS: PTTG1 levels correlate with tumour aggressiveness. PTTG1 silencing causes reduced MTC cell proliferation, supporting the hypothesis that PTTG1 might have an important role in C-cell neoplastic proliferation.

Magmas, a gene newly identified as overexpressed in human and mouse ACTH-secreting pituitary adenomas, protects pituitary cells from apoptotic stimuli.

Pituitary tumors are mostly benign, being locally invasive in 5-35% of cases. Deregulation of several genes has been suggested as a possible alteration underlying the development and progression of pituitary tumors. We here report the identification of a cDNA, corresponding to Magmas gene (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction), which is highly expressed in two different ACTH-secreting mouse pituitary adenoma cell lines as compared with normal pituitary as well as in two thirds of 64 examined pituitary adenomas as compared with human normal pituitary. Tim 16, the mitochondrial protein encoded by Magmas, was indeed expressed in a mouse ACTH-secreting pituitary adenoma cell line, AtT-20 D16v-F2 cells, in a subcellular compartment likely corresponding to mitochondria. Magmas silencing determined a reduced rate of DNA synthesis, an accumulation in G1 phase, and a concomitant decrease in S phase in At-T20 D16v-F2 cells. Moreover, Magmas-silenced cells displayed basal caspase 3/7 activity and DNA fragmentation levels similar to control cells, which both increased under proapoptotic stimuli. Our data demonstrate that Magmas is overexpressed in mouse and human ACTH-secreting pituitary adenomas. Moreover, our results show that Magmas protects pituitary cells from apoptosis, suggesting its possible involvement in neoplastic transformation.

Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids.

Bronchial carcinoids (BCs) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. It has been previously demonstrated that everolimus, or RAD001, an mTOR inhibitor, has potent antiproliferative effects in human endocrine tumors. Our aim was to evaluate the possible antiproliferative effects of everolimus in human BCs in primary culture. We collected 24 BCs that were dispersed in primary cultures, treated without or with 1 nM-1 muM everolimus, 10 nM SOM230 (pasireotide, a somatostatin receptor multiligand), and/or 50 nM IGF1. Cell viability was evaluated after 48 h, and chromogranin A (CgA) as well as vascular endothelial growth factor (VEGF) secretion was assessed after 8 h incubation. Somatostatin receptors, mTOR, and AKT expression were investigated by quantitative PCR. We found that in 15 cultures (67.5%), everolimus significantly reduced cell viability (by approximately 30%; P<0.05 versus control), inhibited p70S6K activity (-30%), and blocked IGF1 proliferative effects. Everolimus also significantly reduced CgA (by approximately 20%) and VEGF (by approximately 15%) secretion. Cotreatment with SOM230 did not exert additive effects on cell viability and secretory activity. AKT expression was similar in responder and nonresponder tissues, while mTOR expression was significantly higher in the responder group, which was characterized by higher CgA plasma levels and bigger tumors with higher mitotic index and angiogenesis. Our data demonstrate that everolimus reduces VEGF secretion and cell viability in BCs with a mechanism likely involving IGF1 signaling, suggesting that it might represent a possible medical treatment for BCs.

Effect of everolimus on cell viability in nonfunctioning pituitary adenomas.

CONTEXT: Pituitary adenomas can cause specific syndromes due to hormone excess and/or determine sellar mass symptoms. Pituitary cell growth can sometimes be influenced by medical therapy, such as for somatotroph adenomas treated with somatostatin analogs or prolactinomas treated with dopaminergic drugs. However, nonfunctioning pituitary adenomas (NFAs) are still orphans of medical therapy. Everolimus (RAD001), a derivative of rapamycin, is a well-known immunosuppressant drug, which has been recently shown to have antineoplastic activity in several human cancers. OBJECTIVE: The objective of the study was to investigate the possible antiproliferative effects of RAD001 in human NFAs. DESIGN: We collected 40 NFAs that were dispersed in primary cultures, treated without or with 1 nm to 1 microm RAD001, 10 nm cabergoline, 10 nm SOM230 (a somatostatin receptor multiligand), and/or 50 nm IGF-I. Cell viability and apoptosis were evaluated after 48 h, and vascular endothelial growth factor (VEGF) secretion was assessed after an 8-h incubation. Somatostatin and dopamine subtype 2 receptor expression was investigated by quantitative PCR. RESULTS: In 28 cultures (70%), Everolimus significantly reduced cell viability (by approximately 40%; P < 0.05 vs. control), promoted apoptosis (+30%; P < 0.05 vs. control), inhibited p70S6K activity (-20%), and blocked IGF-I proliferative and antiapoptotic effects. In selected tissues cotreatment with SOM230, but not cabergoline, exerted an additive effect. Everolimus did not affect VEGF secretion but blocked the stimulatory effects of IGF-I on this parameter. CONCLUSIONS: Everolimus reduced NFA cell viability by inducing apoptosis, with a mechanism likely involving IGF-I signaling but not VEGF secretion, suggesting that it might represent a possible medical treatment of invasive/recurrent NFAs.

BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies.

OBJECTIVE: Papillary thyroid carcinoma (PTC) represents the majority of differentiated thyroid cancers, presenting the V600E activating BRAF mutation in 29-83% of cases. The aim of our study is to analyze the influence of BRAF mutation analysis on the diagnostic accuracy of fine-needle aspiration biopsy (FNAB) in patients with suspected PTC. DESIGN AND METHODS: Thyroid cytoaspirates from 469 nodules (size: 1.1+/-0.8 cm) with ultrasonographic features suspicious of malignant lesion, performed in 374 patients, were submitted to cytological evaluation and to biomolecular analysis, carried out after somatic DNA isolation, specific PCR amplification, and subsequent automated direct sequencing. All PCR fragments were also processed by specific enzyme restriction analysis. RESULTS: BRAF V600E mutation was found in 48 samples, 41 of which were also cytologically diagnosed as PTC, with histologic confirmation after thyroidectomy. Total thyroidectomy was perfomed also in seven patients with negative cytology but positive BRAF mutation, with histological confirmation of PTC in all. Among the 429 BRAF-negative samples, 407 had negative cytology for PTC, while 22 were diagnosed as suspected PTC and underwent total thyroidectomy with histological diagnosis of PTC in 17 and benign lesion in five. The prevalence of BRAF V600E mutation among histologically diagnosed PTC patients was 64%. Biomolecular analysis significantly increased cytology sensitivity for PTC from 77.3 to 86.7% (P<0.01). CONCLUSIONS: These data indicate that BRAF V600E mutation analysis can significantly improve FNAB diagnostic accuracy. However, biomolecular analysis is complementary to cytology, which should always be performed.