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Purpose: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo., Experimental Design: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice., Results: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence., Conclusions: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220., (©2023 American Association for Cancer Research.)

Context: Thyroid cancer recurrence is associated with increased mortality and adverse outcomes. Recurrence risk is currently predicted using clinical tools, often restaging patients after treatment. Detailed understanding of recurrence risk at disease onset could lead to personalized and improved patient care., Objective: We aimed to perform a comprehensive bioinformatic and experimental analysis of 3 levels of genetic change (mRNA, microRNA, and somatic mutation) apparent in recurrent tumors and construct a new combinatorial prognostic risk model., Methods: We analyzed The Cancer Genome Atlas data (TCGA) to identify differentially expressed genes (mRNA/microRNA) in 46 recurrent vs 455 nonrecurrent thyroid tumors. Two exonic mutational pipelines were used to identify somatic mutations. Functional gene analysis was performed in cell-based assays in multiple thyroid cell lines. The prognostic value of genes was evaluated with TCGA datasets., Results: We identified 128 new potential biomarkers associated with recurrence, including 40 mRNAs, 39 miRNAs, and 59 genetic variants. Among differentially expressed genes, modulation of FN1, ITGα3, and MET had a significant impact on thyroid cancer cell migration. Similarly, ablation of miR-486 and miR-1179 significantly increased migration of TPC-1 and SW1736 cells. We further utilized genes with a validated functional role and identified a 5-gene risk score classifier as an independent predictor of thyroid cancer recurrence., Conclusion: Our newly proposed risk model based on combinatorial mRNA and microRNA expression has potential clinical utility as a prognostic indicator of recurrence. These findings should facilitate earlier prediction of recurrence with implications for improving patient outcome by tailoring treatment to disease risk and increasing posttreatment surveillance., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Pancreatic angiosarcoma is an exceedingly rare malignancy accounting for <1% of pancreatic neoplasms. A very limited number of pancreatic angiosarcomas have been reported in the literature without any cases described in children. We present the case of a 17-year-old female diagnosed with angiosarcoma of the pancreas following pancreaticoduodenectomy for a pancreatic mass, initially presumed to be a solid pseudopapillary neoplasm of the pancreas. The angiosarcoma was found to have a novel activating internal tandem duplication in the KDR gene (KDR-internal tandem duplication). We discuss the current literature on this disease process. This is the first reported case of pancreatic angiosarcoma in a pediatric patient and the first with an activating KDR-internal tandem duplication., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The sodium iodide symporter (NIS) functions to transport iodide and is critical for successful radioiodide ablation of cancer cells. Approaches to bolster NIS function and diminish recurrence post-radioiodide therapy are impeded by oncogenic pathways that suppress NIS, as well as the inherent complexity of NIS regulation. Here, we utilize NIS in high-throughput drug screening and undertake rigorous evaluation of lead compounds to identify and target key processes underpinning NIS function. We find that multiple proteostasis pathways, including proteasomal degradation and autophagy, are central to the cellular processing of NIS. Utilizing inhibitors targeting distinct molecular processes, we pinpoint combinatorial drug strategies giving robust >5-fold increases in radioiodide uptake. We also reveal significant dysregulation of core proteostasis genes in human tumors, identifying a 13-gene risk score classifier as an independent predictor of recurrence in radioiodide-treated patients. We thus propose and discuss a model for targetable steps of intracellular processing of NIS function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes-ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)-controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP-a principal component of endoplasmic reticulum (ER)-associated degradation-governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs. SIGNIFICANCE: These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer., (©2019 American Association for Cancer Research.)

Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro , and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. Although the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) predicts poor patient outcome, its mechanisms of action are incompletely understood. We show here that the protein PBF modulates PTTG function, is overexpressed in HNSCC tumors, and correlates with significantly reduced survival. Lentiviral shRNA attenuation of PTTG or PBF expression in HNSCC cells with either wild-type or mutant p53, and with and without HPV infection, led to dysregulated expression of p53 target genes involved in DNA repair and apoptosis. Mechanistically, PTTG and PBF affected each other's interaction with p53 and cooperated to reduce p53 protein stability in HNSCC cells independently of HPV. Depletion of either PTTG or PBF significantly repressed cellular migration and invasion and impaired colony formation in HNSCC cells, implicating both proto-oncogenes in basic mechanisms of tumorigenesis. Patients with HNSCC with high tumoral PBF and PTTG had the poorest overall survival, which reflects a marked impairment of p53-dependent signaling. Significance: These findings reveal a complex and novel interrelationship between the expression and function of PTTG, PBF, and p53 in human HNSCC that significantly influences patient outcome. Cancer Res; 78(20); 5863-76. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The proto-oncogene PTTG and its binding partner PBF have been widely studied in multiple cancer types, particularly thyroid and colorectal, but their combined role in tumourigenesis is uncharacterised. Here, we show for the first time that together PTTG and PBF significantly modulate DNA damage response (DDR) genes, including p53 target genes, required to maintain genomic integrity in thyroid cells. Critically, DDR genes were extensively repressed in primary thyrocytes from a bitransgenic murine model (Bi-Tg) of thyroid-specific PBF and PTTG overexpression. Irradiation exposure to amplify p53 levels further induced significant repression of DDR genes in Bi-Tg thyrocytes (P=2.4 × 10 -4 ) compared with either PBF- (P=1.5 × 10 -3 ) or PTTG-expressing thyrocytes (P=NS). Consistent with this, genetic instability was greatest in Bi-Tg thyrocytes with a mean genetic instability (GI) index of 35.8±2.6%, as well as significant induction of gross chromosomal aberrations in thyroidal TPC-1 cells following overexpression of PBF and PTTG. We extended our findings to human thyroid cancer using TCGA data sets (n=322) and found striking correlations with PBF and PTTG expression in well-characterised DDR gene panel RNA-seq data. In addition, genetic associations and transient transfection identified PBF as a downstream target of the receptor tyrosine kinase-BRAF signalling pathway, emphasising a role for PBF as a novel component in a pathway well described to drive neoplastic growth. We also showed that overall survival (P=1.91 × 10 -5 ) and disease-free survival (P=4.9 × 10 -5 ) was poorer for TCGA patients with elevated tumoural PBF/PTTG expression and mutationally activated BRAF. Together our findings indicate that PBF and PTTG have a critical role in promoting thyroid cancer that is predictive of poorer patient outcome.

Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer., (© 2017 The authors.)

Context: Metastatic disease is responsible for the majority of endocrine cancer deaths. New therapeutic targets are urgently needed to improve patient survival rates., Objective: The proto-oncogene PTTG1-binding factor (PBF/PTTG1IP) is overexpressed in multiple endocrine cancers and circumstantially associated with tumor aggressiveness. This study aimed to understand the role of PBF in tumor cell invasion and identify possible routes to inhibit its action. Design, Setting, Patients, and Interventions: Thyroid, breast, and colorectal cells were transfected with PBF and cultured for in vitro analysis. PBF and cortactin (CTTN) expression was determined in differentiated thyroid cancer and The Cancer Genome Atlas RNA-seq data., Primary Outcome Measure: Pro-invasive effects of PBF were evaluated by 2D Boyden chamber, 3D organotypic, and proximity ligation assays., Results: Our study identified that PBF and CTTN physically interact and co-localize, and that this occurs at the cell periphery, particularly at the leading edge of migrating cancer cells. Critically, PBF induces potent cellular invasion and migration in thyroid and breast cancer cells, which is entirely abrogated in the absence of CTTN. Importantly, we found that CTTN is over-expressed in differentiated thyroid cancer, particularly in patients with regional lymph node metastasis, which significantly correlates with elevated PBF expression. Mutation of PBF (Y174A) or pharmacological intervention modulates the PBF: CTTN interaction and attenuates the invasive properties of cancer cells., Conclusion: Our results demonstrate a unique role for PBF in regulating CTTN function to promote endocrine cell invasion and migration, as well as identify a new targetable interaction to block tumor cell movement.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERβ expression in the MTC tumour. ERα and ERβ form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERβ represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors., (© 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.)

Unlabelled: Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability., Significance: Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors., (©2015 American Association for Cancer Research.)

The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

Context: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient., Objective: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors., Results: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells., Conclusions: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

The pituitary tumor transforming gene (PTTG) is a multifunctional proto-oncogene that is over-expressed in various tumors including thyroid carcinomas, where it is a prognostic indicator of tumor recurrence. PTTG has potent transforming capabilities in vitro and in vivo, and many studies have investigated the potential mechanisms by which PTTG contributes to tumorigenesis. As the human securin, PTTG is involved in critical mechanisms of cell cycle regulation, whereby aberrant expression induces aneuploidy. PTTG may further contribute to tumorigenesis through its role in DNA damage response pathways and via complex interactions with hormones and growth factors. Furthermore, PTTG over-expression negatively impacts upon the efficacy of radioiodine therapy in thyroid cancer, through repression of expression and function of the sodium iodide symporter. Given its various roles at all disease stages, PTTG appears to be an important oncogene in thyroid cancer. This review discusses the current knowledge of PTTG with particular focus on its role in thyroid cancer.

Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.

Pituitary tumor-transforming gene (PTTG)-binding factor (PBF; PTTG1IP) was initially identified through its interaction with the human securin, PTTG. Like PTTG, PBF is upregulated in multiple endocrine tumours including thyroid cancer. PBF is believed to induce the translocation of PTTG into the cell nucleus where it can drive tumourigenesis via a number of different mechanisms. However, an independent transforming ability has been demonstrated both in vitro and in vivo, suggesting that PBF is itself a proto-oncogene. Studied in only a limited number of publications to date, PBF is emerging as a protein with a growing repertoire of roles. Recent data suggest that PBF possesses a complex multifunctionality in an increasing number of tumour settings. For example, PBF is upregulated by oestrogen and mediates oestrogen-stimulated cell invasion in breast cancer cells. In addition to a possible role in the induction of thyroid tumourigenesis, PBF overexpression in thyroid cancers inhibits iodide uptake. PBF has been shown to repress sodium iodide symporter (NIS) activity by transcriptional regulation of NIS expression through the human NIS upstream enhancer and further inhibits iodide uptake via a post-translational mechanism of NIS governing subcellular localisation. This review discusses the current data describing PBF expression and function in thyroid cancer and highlights PBF as a novel target for improving radioiodine uptake and thus prognosis in thyroid cancer.

The pituitary tumor-transforming gene (PTTG1) encodes a multifunctional protein (PTTG) that is overexpressed in numerous tumours, including pituitary, thyroid, breast and ovarian carcinomas. PTTG induces cellular transformation in vitro and tumourigenesis in vivo, and several mechanisms by which PTTG contributes to tumourigenesis have been investigated. Also known as the human securin, PTTG is involved in cell cycle regulation, controlling the segregation of sister chromatids during mitosis. This review outlines current information regarding PTTG structure, expression, regulation and function in the pathogenesis of neoplasia. Recent progress concerning the use of PTTG as a prognostic marker or therapeutic target will be considered. In addition, the PTTG binding factor (PBF), identified through its interaction with PTTG, has also been established as a proto-oncogene that is upregulated in several cancers. Current knowledge regarding PBF is outlined and its role both independently and alongside PTTG in endocrine and related cancers is discussed.

Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects., ((c)2010 AACR.)

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T(3) uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T(3) transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T(3) and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T(3) uptake. Given that T(3) stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T(3) transport, mediated through the modulation of cell proliferation in the developing brain.

Objective: To identify the type 2 diabetes gene located at chromosome 18p11., Research Design and Methods: We investigated the region in a young genetically isolated population by genotyping 34 single nucleotide polymorphisms (SNPs) in 78 case subjects and 101 control subjects. Two SNPs were selected and followed up in two cohorts. The first cohort came from a general Dutch population. In this cohort, association with type 2 diabetes was investigated using 616 type 2 diabetic case subjects and 2,890 control subjects; association with oral glucose tolerance test data was performed in 361 normoglycemic people. Association with fat distribution was studied in the second replication cohort, consisting of 836 people from the genetically isolated population., Results: At the initial step, we found that the common C allele of SNP rs3745012 was associated with type 2 diabetes (odds ratio 2.01, P = 0.03). This SNP is located at the 3' untranslated region of the LPIN2 gene, which is a plausible candidate for type 2 diabetes and obesity. In the cohort from the general Dutch population, we demonstrated that rs3745012 interacts with BMI in determination of type 2 diabetes: whereas in subjects with high BMI, the common C allele is associated with type 2 diabetes, the same allele exhibits a neutral or protective effect in lean subjects (P = 0.05 overall effect, P = 0.02 interaction). Most remarkably, rs3745012 strongly affected composite insulin sensitivity index (P = 0.006 for overall effect, P = 0.004 for interaction). In the second replication cohort, we found that the allele C of rs3745012 increases trunk-to-legs fat mass ratio (P = 0.001) and may affect other fat-related measurements., Conclusions: rs3745012 SNP of the LPIN2 gene is associated with type 2 diabetes and fat distribution.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Genetic instability (GI) is a hallmark feature of tumor development. Securin, also known as pituitary tumor transforming gene (PTTG), is a mitotic checkpoint protein which is highly expressed in numerous cancers, is associated with tumor invasiveness, and induces GI in thyroid cells. We used fluorescence inter-simple sequence repeat PCR to assess GI caused primarily by DNA breakage events in 19 colorectal tumors. GI values ranged significantly, with Dukes' stage C&D colorectal tumors exhibiting greater GI and higher securin expression than Dukes' stage A&B tumors. Consistent with these findings, we observed a dose-dependent increase in GI in HCT116 cells in response to securin overexpression, as well as in non-transformed human fibroblasts. As securin has been implicated in a novel DNA repair pathway in fission yeast, we investigated its potential role in chemotoxic DNA damage response pathways in mammalian cells, using host cell reactivation assays. Securin overexpression in HCT116 cells inhibited etoposide-induced double-stranded DNA damage repair activity, and repressed Ku heterodimer function. Additionally, we observed that securin and Ku70 showed a reciprocal cytosol-nuclear translocation in response to etoposide-induced dsDNA damage. Our data suggest that, by repressing Ku70 activity and inhibiting the non-homologous end-joining dsDNA repair pathway, securin may be a critical gene in the development of GI in colorectal cancer.

The G1P1A[8] rotavirus vaccine candidate 89-12, the precursor to Rotarix, stimulated high titers of neutralizing antibodies to non-G1/P1A[8] serotypes of human rotavirus in naturally infected subjects before attenuation by cell-culture passages. These responses were greatly diminished in young infants (median age, 11 weeks) administered the attenuated vaccine. Because of the possibility of improved responses in older infants, the immunogenicity of the 89-12 vaccine candidate was evaluated after administration of 2 doses beginning at either 4 or 6 months of age. As was found in young infants, neutralizing antibody responses to non-G1/P1A[8] rotaviruses were considerably lower than those observed after natural infection. The reasons identified were overall (P<.0001) lower neutralizing antibody responses stimulated by the attenuated 89-12 strain, compared with those stimulated by its virulent precursor, and 5 mutations selected in the gene encoding the immunodominant VP4 (P) neutralization protein. Even so, the Rotarix vaccine developed from attenuated 89-12 was found to elicit excellent protection against non-G1 rotaviruses.

A quadrivalent precursor to the pentavalent rotavirus vaccine candidate RotaTeq was evaluated in a 3-dose, 439-subject study. To determine immunogenicity, the quantity of rotavirus immunoglobulin A (IgA) in stool specimens obtained, at 1 of 10 study sites, from 37 placebo and 37 vaccine recipients was measured. None of the placebo recipients showed a clinically important (>/=3-fold) increase in stool rotavirus IgA, whereas 31 vaccine recipients showed an increase after at least 1 dose of vaccine. In total, 16, 19, and 15 vaccine recipients had increases after 1, 2, and 3 doses, respectively, indicating that a 3-dose regimen increased the immune response elicited by this vaccine.

Excess coronary heart disease morbidity and mortality among African Americans remains an important yet unexplained public health problem. We hypothesized that adverse outcome is in part due to intrinsic or acquired abnormalities in coronary endothelial function and vasoreactivity. We compared dose-response curves relating changes in coronary blood flow and epicardial diameter to graded infusions of acetylcholine in 50 African American and 65 white subjects with hypertensive left ventricular hypertrophy (LVH) and normal coronary arteries. These groups were similar for age, body mass index, mean arterial pressure, and indexed left ventricular mass. The same protocol was conducted in 24 normotensive African American and 56 similar white subjects. We found significant depression in the coronary blood flow dose-response curve relation among African Americans when compared with white subjects with similar LVH (P<0.03). Racial differences were observed at all doses of acetylcholine but were less precisely estimated at the highest dose. The same testing among normotensive subjects revealed similar dose-response curves with no significant effect of race. Qualitatively similar results were found with respect to coronary diameter. Adenosine responses, a measure of endothelium-independent function, were similar after partitioning by LVH. Our study demonstrates that there are racial differences in sensitivity of coronary arteries to acetylcholine-stimulated relaxation among those with LVH. These results provide a mechanism whereby racial differences in coronary vasoreactivity might contribute to adverse coronary heart disease outcome among African Americans, a group in whom LVH is prevalent.

Objectives: The purpose of our study was to determine if the presence of African American ethnicity modulates improvement in coronary vascular endothelial function after supplementary L-arginine., Background: Endothelial dysfunction is an early stage in the development of coronary atherosclerosis and has been implicated in the pathogenesis of hypertension and cardiomyopathy. Amelioration of endothelial dysfunction has been demonstrated in patients with established coronary atherosclerosis or with risk factors in response to infusion of L-arginine, the precursor of nitric oxide. Racial and gender patterns in L-arginine responsiveness have not, heretofore, been studied., Methods: Invasive testing of coronary artery and microvascular reactivity in response to graded intracoronary infusions of acetylcholine (ACh) +/- L-arginine was carried out in 33 matched pairs of African American and white subjects with no angiographic coronary artery disease. Pairs were matched for age, gender, indexed left ventricular mass, body mass index and low-density lipoprotein cholesterol., Results: In addition to the matching parameters, there were no significant differences in peak coronary blood flow (CBF) response to intracoronary adenosine or in the peak CBF response to ACh before L-arginine infusion. However, absolute percentile improvement in CBF response to ACh infusion after L-arginine, as compared with before, was significantly greater among African Americans as a group (45 +/- 10% vs. 4 +/- 6%, p = 0.0016) and after partitioning by gender. The mechanism of this increase was mediated through further reduction in coronary microvascular resistance. L-arginine infusion also resulted in greater epicardial dilator response after ACh among African Americans., Conclusions: We conclude that intracoronary infusion of L-arginine provides significantly greater augmentation of endothelium-dependent vascular relaxation in those of African American ethnicity when compared with matched white subjects drawn from a cohort electively referred for coronary angiography. Our findings suggest that there are target populations in which supplementary L-arginine may be of therapeutic benefit in the amelioration of microvascular endothelial dysfunction. In view of the excess prevalence of cardiomyopathy among African Americans, pharmacologic correction of microcirculatory endothelial dysfunction in this group is an important area of further investigation and may ultimately prove to be clinically indicated.

Previous studies have identified a number of echocardiographic variables that predict cardiovascular disease (CVD) events and mortality, but have not focused on a large elderly cohort. The purpose of this study was to determine whether M-mode echocardiographic variables predicted all-cause mortality, incident coronary heart disease (CHD), congestive heart failure (CHF), and stroke in a large prospective, multicenter, population-based study. In the Cardiovascular Health Study, a biracial cohort of 5,888 men and women (mean age 73 years) underwent 2-dimensional M-mode echocardiographic measurements of left ventricular (LV) internal dimensions, wall thickness, mass and geometry, as well as measurement of left atrial dimension and assessment for mitral annular calcium. Participants were followed for 6 to 7 years for incident events; analyses excluded subjects with prevalent disease. One or more echocardiographic measurements were independent predictors of all-cause mortality and incident CHD, CHF, and stroke. After adjustment for anthropometric and traditional CVD risk factors, LV mass was significantly related to incident CHD, CHF, and stroke. The highest quartile of LV mass conferred a hazards ratio of 3.36, compared with the lowest quartile, for incident CHF. Furthermore, incident CHF-free survival was significantly lower for participants with LV mass in the highest versus the 2 lowest quartiles (86% vs 97%, respectively, at 2,500 days). Eccentric and concentric LV hypertrophy, respectively, conferred adjusted hazards ratios, compared with normal LV geometry, of 2.05 and 1.61 for incident CHD, and 2.95 and 3.32 for incident CHF. Thus, in an elderly biracial population, selected 2-dimensional M-mode echocardiographic measurements were important markers of subclinical disease and conferred independent prognostic information for incident CVD events, especially CHF and CHD.

This manuscript describes the results of invasive testing of renal artery and arteriolar reactivity in two patients using a panel of endothelium-dependent and -independent agents including radiographic contrast medium. We found that the renal artery and microcirculation dilate in response to graded acetylcholine infusions and to bolus nitroglycerin infusion; the renal microcirculation dilates in response to papaverine but constricts after adenosine and after radiographic contrast medium. Future indications for this testing are briefly discussed.

Background: Rotavirus is the most common cause of severe, dehydrating diarrhoea in infants worldwide. We assessed the safety, immunogenicity, and efficacy of a live, oral human rotavirus vaccine, 89-12, in US children in a randomised, placebo-controlled, double-blind multicentre trial., Methods: 215 healthy infants were enrolled, of whom 213 were given two doses of 89-12 (containing 1x10(5) plaque-forming units) or placebo, and 213 were followed up through one rotavirus season. The frequency of side-effects was compared for 7 days after each dose of vaccine. Immune responses to rotavirus were assessed by serum and stool IgA, and by serum 89-12 neutralising titres. The primary outcome variable (protection from rotavirus disease) was evaluated by comparing the frequencies of rotavirus gastroenteritis in an intention-to-treat analysis., Findings: Adverse reactions were mild. Low-grade fever (> or = 38.1 degrees C) after the first dose was the only side-effect significantly more common in the vaccine group than in the placebo group (21 [19%] vs 5 [5%], p=0.001). An immune response to vaccine was detected in 94.4% of vaccinees. Rotavirus disease occurred in 18 of 107 placebo recipients and two of 108 vaccine recipients (vaccine efficacy 89.0% [95% CI 65.4-94.5]). Ten infants in the placebo group but none in the vaccine group were presented for medical care., Interpretation: The 89-12 rotavirus vaccine was safe and immunogenic and provided a high degree of protection against rotavirus disease. Further investigations of this vaccine are needed to confirm these findings in other settings.

Changes in left ventricular (LV) diastolic function (e.g., as measured by transmitral flow velocity) are known to occur with aging. In addition, impaired LV diastolic function plays an important role in such cardiovascular disorders common in the elderly as hypertension, ischemic heart disease, and congestive heart failure (CHF). Participants in the Cardiovascular Health Study, a multicenter study of community-dwelling men (n=2,239) and women (n=2,962) > or = 65 years of age, underwent an extensive baseline evaluation, including echocardiography. Early diastolic LV Doppler (transmitral) peak filling velocity decreased, and peak late diastolic (atrial) velocity increased with age in multivariate analyses (all p <0.001). Early and late diastolic peak filling velocities were both significantly higher in women than in men, even after adjustment for body surface area (or height and weight). In multivariate models in the entire cohort and a healthy subgroup (n=703), gender, age, heart rate, and blood pressure (BP) were most strongly related to early and late diastolic transmitral peak velocities. Early and late diastolic peak velocities both increased with increases in systolic BP and decreased with increases in diastolic BP (p <0.001). Doppler transmitral velocities were compared among health status subgroups. In multiple regression models adjusted for other covariates, and in analysis of variance models examining differences across subgroups adjusted only for age, the subgroup with CHF had the highest early diastolic peak velocities. All clinical disease subgroups had higher late diastolic peak velocities than the healthy subgroup, with the subgroups with either CHF or hypertension having the highest age-adjusted means. The subgroup with hypertension had the lowest ratio of early-to-late diastolic peak velocity, and men with CHF had the highest ratio. These findings are consistent with previous reports that hypertensive subjects exhibit an abnormal relaxation pattern, whereas patients with CHF develop a pattern suggestive of an increased early diastolic left atrial-LV pressure gradient.

The safety and immunogenicity of an orally administered live human rotavirus vaccine candidate (89-12), attenuated by 33 passages in monkey kidney cells, were evaluated in placebo-controlled trials in adults, children and infants. This strain was selected because natural infections with 89-12-like rotaviruses provided 100% protection over two years. The initial evaluations in adults, seropositive children and nine infants indicated that the vaccine was safe. Two doses of vaccine (10(5) p.f.u. dose-1) or placebo were then given to 42 infants, aged from 6 to 26 weeks. No significant difference in side effects was seen. Seroconversion was demonstrated in 19 of 20 previously uninfected vaccine recipients, but > or = 4-fold rises in 89-12 neutralizing antibody titers were detected in only seven subjects. Intestinal IgA responses were detected in 15 subjects. This attenuated human rotavirus was safe and immunogenic and should be further evaluated as a vaccine candidate.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.

Objectives: The aim of this study was to determine the prevalence of aortic sclerosis and stenosis in the elderly and to identify clinical factors associated with degenerative aortic valve disease., Background: Several lines of evidence suggest that degenerative aortic valve disease is not an inevitable consequence of aging and may be associated with specific clinical factors., Methods: In 5,201 subjects > or = 65 years of age enrolled in the Cardiovascular Health Study, the relation between aortic sclerosis or stenosis identified on echocardiography and clinical risk factors for atherosclerosis was evaluated by using stepwise logistic regression analysis., Results: Aortic valve sclerosis was present in 26% and aortic valve stenosis in 2% of the entire study cohort; in subjects > or = 75 years of age, sclerosis was present in 37% and stenosis in 2.6%. Independent clinical factors associated with degenerative aortic valve disease included age (twofold increased risk for each 10-year increase in age), male gender (twofold excess risk), present smoking (35% increase in risk) and a history of hypertension (20% increase in risk). Other significant factors included height and high lipoprotein(a) and low density lipoprotein cholesterol levels., Conclusions: Clinical factors associated with aortic sclerosis and stenosis can be identified and are similar to risk factors for atherosclerosis.

Excess cardiovascular morbidity and mortality among African (black) Americans remains an important yet unexplained public health problem. One possible explanation proposes that intrinsic or acquired abnormalities in coronary vascular reactivity and endothelial function result in excess ischemia among black Americans. To examine this hypothesis, we subjected 80 individuals with normal coronary arteries to invasive testing of coronary artery and microvascular relaxation using intracoronary infusions of acetylcholine and adenosine, a Doppler tipped intracoronary guide wire, and quantitative coronary angiography. We measured the percent increase in coronary blood flow and epicardial diameter after graded infusion of intracoronary acetylcholine and in coronary blood flow after intracoronary adenosine in 31 normotensive subjects (10 black, 21 white) and 49 hypertensive subjects with left ventricular hypertrophy (25 black, 24 white). Categorical and multivariate analyses revealed that in response to intracoronary adenosine and acetylcholine, the depression in endothelium-independent and -dependent microvascular relaxation during peak agonist effect was largely related to the presence of chronic hypertension and left ventricular hypertrophy. Normotensive subjects demonstrated no intrinsic racial differences in conduit and resistance vessel vasoreactivity. In response to maximal infusion of acetylcholine, epicardial coronary arteries constricted similarly in black and white subjects with hypertensive left ventricular hypertrophy and dilated similarly in normotensive black and white subjects. Thus, our study shows that in a cohort of black and white subjects referred for coronary arteriography because of chest pain, African American race is not associated with excess intrinsic or acquired depression in coronary vascular relaxation during the peak effect of the endothelium-dependent and -independent agonists acetylcholine and adenosine, after adjustment for the presence of left ventricular hypertrophy.

Background and Objectives: Excess cardiovascular morbidity and mortality among African (black) Americans is the subject of intensive investigation but the etiology remains speculative. One hypothesis proposes that inherent, or intrinsic, differences in coronary vascular reactivity and endothelial function predispose African Americans to enhanced vasoconstriction and/or depressed vasodilation, resulting in excess ischemia. The objective of this study was to establish whether coronary vasoreactivity differs among normotensive, nondiabetic African and white Americans with normal arteries referred for coronary arteriography because of chest pain., Patients and Methods: Eleven African American (8 female, 3 male) and 28 white American (9 female, 19 male) normotensive, euglycemic patients with normal coronary arteries were prospectively recruited for invasive testing of coronary artery and microvascular relaxation using the endothelium-dependent and -independent agents, acetylcholine and adenosine; a Doppler tipped intracoronary guidewire; and quantitative coronary angiography., Results: The study cohort consisted of 17 women (44%) and 22 men (56%) with a mean age of 46 +/- 10 yrs. Of 8 African American women, 6 were premenopausal and 2 were postmenopausal on estrogen replacement therapy. Of 9 white American women, 2 were premenopausal, 1 was 46-year old with a previous history of hysterectomy without ovariectomy, 2 were postmenopausal on estrogen replacement therapy, 2 were perimenopausal and 44- and 54-year old, and 2 were postmenopausal without estrogen replacement therapy. In response to maximal infusion of acetylcholine, epicardial coronary arteries and resistance vessels dilated similarly in black and white subjects. Dose-response curves revealed no significant racial differences during submaximal graded infusion of acetylcholine. In response to peak effect of adenosine, there were no racial differences in dilation of the microcirculation., Conclusions: In the absence of hypertension, diabetes mellitus, and angiographic evidence of coronary artery disease, African American women demonstrate no evidence of intrinsic predisposition to enhanced coronary conduit vasoconstriction or depressed microcirculatory dilation in response to the endothelium-dependent and -independent vasodilator agonists-acetylcholine and adenosine-when compared with responses of similar white men and women. Because of low enrollment of black males, definitive conclusions cannot be drawn regarding this group.

This report describes the relation among diabetes, blood pressure, and prevalent cardiovascular disease, and echocardiographically measured left ventricular mass and filling (transmitral valve flow) velocities in the Cardiovascular Health Study, a cohort of 5201 men and women > or = 65 years of age. Ventricular septal and left posterior wall thicknesses were greater in diabetic than in nondiabetic subjects, showing a significant linear trend (p = 0.025 for ventricular septal thickness in both sexes combined, p = 0.002 for posterior wall thickness) with increased duration of diabetes. Increased wall thickness of the ventricular septum or the left posterior wall was not associated with prevalent coronary heart disease (CHD) in the cohort. Increased left ventricular mass was associated with diabetic persons not reporting CHD and with all subjects with CHD regardless of glucose tolerance status. After adjusting for body weight, blood pressure, heart rate, and prevalent coronary or cerebrovascular disease, diabetes (as measured by glucose level, insulin use, oral hypoglycemic use, and a positive history of diabetes before baseline examination) remained an independent predictor of increased left ventricular mass among men and women (174.2 gm in diabetic men vs 169.8 gm in normal men, 138.2 gm in diabetic women vs 134.0 gm in normal women, p = 0.043 for both sexes combined). Both early and late diastolic transmitral peak flow velocities were higher with increased duration of diabetes, but the calculated ratio of the early peak flow velocity to the late velocity (E/A ratio) did not differ significantly between subjects with historical diabetes and those with normal fasting glucose (both genders combined, p = 0.190). Glucose level, insulin use, oral hypoglycemic use, and a positive history of diabetes before baseline examination were significant independent predictors of the late transmitral peak flow velocity and its integrated flow-velocity curve but not for the integral of the early peak flow velocity or the E/A ratio. Diabetes is associated with abnormal left ventricular structure and function in elderly persons. This association persists after adjustment for body weight, blood pressure, heart rate, and reported coronary or cerebrovascular disease.

In a referral normal cardiac population, endothelium-independent coronary relaxation is nearly always normal, but endothelium-dependent relaxation may be depressed in a significant proportion of patients. Further study of the natural history of referral subjects with endothelial dysfunction is necessary to assess the potential cardiovascular risk of this finding in a presumed low-risk population.

Associations of carotid artery diameter and intimal-medial thickness by ultrasound with echocardiographic left ventricular (LV) structure were examined in 3,409 participants in the Cardiovascular Health Study, a population-based study of risk factors for coronary heart disease and stroke in men and women aged > or = 65 years. At baseline, sector-guided M-mode echocardiography and B-mode ultrasound were used to evaluate the left ventricle and carotid arteries, respectively. Common carotid artery diameter and intimal-medial thickness were significantly related to LV mass in correlational analysis (r=0.40 and 0.20, respectively, p<0.01), and each was independently associated with LV mass after adjustment for age, gender, weight, systolic and diastolic blood pressure, antihypertensive medication use, prior coronary heart disease, electrocardiographic abnormalities, high-density lipoprotein, and factor VII. We speculate that changes in the arterial wall affect impedance to LV ejection leading to increases in LV mass. Further follow-up of this cohort is in progress and will help to determine whether such carotid artery measures could, by exacerbating LV hypertrophy, constitute another important risk factor for adverse cardiovascular outcomes.

Background: Left ventricular (LV) hypertrophy, as measured by M-mode echocardiography, is an independent predictor of mortality and/or morbidity from coronary heart disease (CHD). LV global and segmental systolic dysfunction also have been associated with myocardial ischemia and cardiovascular morbidity and mortality. Echocardiographic data, especially two-dimensional, have not been available previously from multicenter-based studies of the elderly. This report describes the distribution and relation at baseline of echocardiographic LV mass and global and segmental LV wall motion to age, sex, and clinical disease category in the Cardiovascular Health Study (CHS), a cohort of 5201 men and women (4850 white) 65 years of age and older., Methods and Results: M-mode LV mass adjusted for body weight increased modestly with age (P < .0001), increasing less than one gram per year increase in age for both men and women. After adjustment for weight, LV mass was significantly greater in men than in women and in participants with clinical CHD compared with participants with neither clinical heart disease nor hypertension (both P < .001). Across all CHS age subgroups, the difference in weight-adjusted LV mass by sex was greater in magnitude than the difference related to clinical CHD. M-mode measurements of LV mass could not be made in 34% of CHS participants, and this was highly related to age (29% in the 65 to 69 year versus 50% in the 85+ year age group, P < .001) and other risk factors. In participants with clinical CHD and with neither clinical heart disease nor hypertension, LV ejection fraction and segmental wall motion abnormalities were more prevalent in men than women (all P < .001). Of interest, 0.5% of men and 0.4% of women with neither clinical heart disease nor hypertension had LV segmental wall motion abnormalities, suggesting silent disease, compared with 26% of men and 10% of women in the clinical CHD group (P < .0001). Multivariate analyses revealed male sex and presence of clinical CHD (both P < .001) to be independent predictors of LV akinesis or dyskinesis., Conclusions: Significant baseline relations were detected between differences in sex, prevalent disease status, and echocardiographic measurements of LV mass and systolic function in the CHS cohort. Age was weakly associated with LV mass measurements and LV ejection fraction abnormalities. These relations should be considered in evaluating the preclinical and clinical effects of CHD risk factors in the elderly.

The Cardiovascular Health Study provided the opportunity to determine the association of subclinical and clinical cardiovascular disease with pulmonary function in a population sample of elderly adults. Included were 2,955 women and 2,246 men over age 64 years who were recruited for this observational study from four communities and completed extensive examinations that included spirometry, echocardiograms, and blood pressure. Current smokers, past smokers with >20 pack-years of smoking, and persons with a history of asthma, chronic bronchitis, or emphysema were excluded from this analysis, leaving 2,784 (55%) of the cohort. Systolic hypertension or coronary artery disease was associated with 40- to 100-mL decrements in FEV1, and 50- to 150-mL decrements in FVC, while a history of congestive heart failure was associated with 200 to 300 mL lower FEV1 and FVC values (p < 0.0001), after correcting for age, height, and waist size. Higher left ventricular (LV) mass was also significantly associated with a decrease in FEV1 and FVC in multivariate models. This relationship was strongest with the end-diastolic LV posterior wall thickness component of LV mass. In summary, FEV1 and FVC are reduced in elderly persons with hypertension, ischemic heart disease, higher disease, higher LV mass, and congestive heart failure, though the magnitude of these associations is relatively small unless heart failure supervenes. Substantial decrements in percent predicted FEV1 and FVC should not be attributed to the presence of uncomplicated ischemic heart disease or hypertension alone.