Anoctamin 1 is Apically Expressed on Thyroid Follicular Cells and Contributes to ATP- and Calcium-Activated Iodide Efflux

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Anoctamin 1 is Apically Expressed on Thyroid Follicular Cells and Contributes to {ATP}- and Calcium-Activated Iodide Efflux

Background/Aims: Iodide efflux from thyroid cells into the follicular lumen is essential for the synthesis of thyroid hormones, however, the pathways mediating this transport have only been partially identified. A calcium-activated pathway of iodide efflux has long been recognized, but its molecular identity unknown. Anoctamin 1 (ANO1) is a calcium activated chloride channel (CaCC), and this study aims to investigate its contribution to iodide fluxes in thyroid cells. Methods: RT-PCR, immunohistochemistry, and live cell imaging with the fluorescent halide biosensor YFP-H148Q/I152L were used to study the expression, localization and function of ANO1 in thyroid cells. Results: ANO1 mRNA was detected in human thyroid tissue and FRTL-5 thyrocytes, and ANO1 protein was localized to the apical membrane of follicular cells. ATP induced a transient loss of iodide from FRTL-5 cells that was dependent on the mobilization of intracellular calcium, and was inhibited by CaCC/ANO1 inhibitors and si RNA against ANO1. Calcium activated iodide efflux was also observed in CHO cells over expressing the Sodium Iodide Symporter (NIS) and ANO1. Conclusion: ANO1 in thyrocytes functions as a calcium activated channel mediating iodide efflux, and may contribute to the rapid delivery of iodide into the follicular lumen for the synthesis of thyroid hormones following activation by calcium-mobilizing stimuli. Copyright (C) 2014 S. Karger AG, Base

Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing

<div><p>Background</p><p>Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for accurate detection of high-grade cervical lesions and cancer. Methylation status has been widely investigated for HPV16, sporadically for other HPV types.</p><p>Methods</p><p>Objective of this methodological study was to set up molecular methods to test the methylation levels in the twelve oncogenic HPV types by pyrosequencing, minimizing the number of HPV type-specific PCR protocols. Target CpGs were selected on the HPV L1 (two regions, L1 I and L1 II) and L2 genes.</p><p>Study samples included DNA stored at Turin, Italy, purified by cervical cells collected in Standard Transport Medium or PreservCyt from women who participated in two studies (N = 126 and 140) nested within the regional organized screening programme.</p><p>PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types.</p><p>Results</p><p>Generation of consensus primers was successful for L1 I and II regions, unsuccessful for L2 region, for which HPV type-specific primers remained necessary. The difference between replicated tests on the same sample was ≤4% in 88%, 77% and 91% of cases when targeting the L1 I, L1 II and L2 regions, respectively. The corresponding intra-class correlation coefficients (ICC) were 0.94, 0.87 and 0.97 respectively. When comparing methylation measures based on consensus and type-specific primers, ICC was 0.97 for the L1 I region and 0.99 the for L1 II region.</p><p>Conclusions</p><p>The proposed protocols, applying consensus primers suitable to amplify the oncogenic HPV types and minimize the number of PCR reactions, represent a promising tool to quantify viral methylation in women positive for any high risk HPV type.</p><p>Impact</p><p>Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.</p></div

Correlation of the percentage of methylation between methylation protocols using consensus or type specific primers.

<p>Correlation of the percentage of methylation between methylation protocols using consensus or type specific primers.</p

L2. Primers sequences and annealing temperatures.

<p>L2. Primers sequences and annealing temperatures.</p

Delta methylation percentage between replicates.

<p>The same bisulfite modified DNA samples (N = 140) were used to test methylation on HPV L1 I, L1 II and L2 regions. For L2 regions only 105 had sufficient material for replicates.</p

Reference sequences for the twelve oncogenic HPV types and CpG positions.

<p>Reference sequences for the twelve oncogenic HPV types and CpG positions.</p

Correlation of the percentage of methylation between the two replicates for each evaluated hrHPV region.

<p>The same bisulfite modified DNA samples (N = 140) were used to test methylation on HPV L1 I, L1 II and L2 regions. For L2 regions only 105 had sufficient material for replicates.</p

Interobserver reproducibility of cytologic p16INK4a/Ki-67 dual immunostaining in human papillomavirus-positive women

BACKGROUND: The accumulation of cyclin-dependent kinase inhibitor 2A (p16(ink4a)) protein in a cell is associated with neoplastic progression in precancerous cervical lesions. Dual staining for p16(ink4a) and Ki-67 has been proposed as a triage test in cervical cancer screening for women who test positive for human papillomavirus DNA. In this study, interobserver reproducibility of the interpretation of this test was assessed. METHODS: Forty-two immunostained, liquid-based cytology slides were divided into 2 sets and were interpreted by 17 to 21 readers from 9 different laboratories, yielding a total of 816 reports. Immunostaining results were classified as positive, negative, inconclusive, or inadequate. After evaluation of the first set of slides and before circulation of the second set, the results were discussed in a plenary meeting. The 10 slides with the most discordant results were evaluated again by selected expert cytopathologists. RESULTS: The overall kappa value was 0.612 (95% confidence interval [CI], 0.523-0.701), it was higher for the positive and negative categories (kappa=0.692 and kappa=0.641, respectively), and it was almost null for the inconclusive category (kappa=0.058). Considering only readers from laboratories with documented experience, the kappa value was higher (kappa=0.747; 95% CI, 0.643-0.839) compared with nonexperienced centers (kappa=0.498; 95% CI, 0.388-0.616). The results were similar in both sets of slides (kappa=0.505 [95% CI, 0.358-0.642] and kappa=0.521 [95% CI, 0.240-0.698] for the first and second sets, respectively). Reinterpretation of the slides with the most discordant results did not provide any improvement (first evaluation, kappa=0.616 [95% CI, 0.384-0.866]; second evaluation, kappa=0.403 [95% CI, 0.182-0.643]). CONCLUSIONS: Dual staining for p16(ink4a) and Ki-67 demonstrated good reproducibility, confirming its robustness, which is a necessary prerequisite for its adoption as a triage test in cervical cancer screening programs that use human papillomavirus DNA as a primary test. Cancer Cytopathol 2017; 125:212-20. (C) 2016 American Cancer Society