Human respiratory syncytial virus (HRSV) is the leading cause of hospitalization of children aged <5 years due to respiratory illness in industrialized countries, and pneumonia is the leading cause of mortality among children aged <5 years worldwide. Although HRSV was first identified in 1956, a preventative vaccine has yet to be developed. Here we report the results of the first study to investigate the circulation and genetic diversity of HRSV in Cambodia among an all-ages population over 5 consecutive years. The incidences of HRSV infection among all-ages outpatient and hospitalized populations were equivalent, at 9.5% and 8.2%, respectively. Infection was most prevalent among children aged <5 years, with bronchiolitis being the most frequently observed clinical syndrome in the same age group. Circulation of HRSV was seasonal, typically coinciding with the rainy season between July and November annually. Strains belonging to HRSV groups A and B were detected with equivalent frequencies; however, we observed a potentially biennial shift in the predominant circulating HRSV genotype. The majority of HRSV group B strains belonged to the recently described BA genotype, with the exception of 10 strains classified as belonging to a novel HRSV group B genotype, SAB4, first reported here.

Estrogen receptor (ER) β is the predominant ER in granulosa cells of the ovary. ERβ is expressed at high levels in granulosa cell tumors (GCT) and in the human GCT-derived cell lines, COV434 and KGN. To gain insight into ERβ function in granulosa cells and in GCT, we have used the COV434 and KGN cell lines. Although the cells bind estradiol (E₂), transcriptional activation of a transfected estrogen-responsive reporter vector construct (ERE₂-luc) by E₂ was not observed. Transactivation was also not observed with cotransfected ERα or β. This transcriptional resistance is specific to steroid receptor transactivation; reporter plasmids that are activated by the transcription factors activator protein 1 (AP-1) and nuclear factor κB (NF-κB) demonstrate both constitutive and inducible transactivation. AP-1 and NF-κB are known to cause transrepression of both ERα- and glucocorticoid receptor-mediated transcription. We therefore examined the possibility that activation of these pathways was responsible for the lack of a response to estrogen by using inhibitors of AP-1 or NF-κB. The AP-1 inhibitors alone had no effect, whereas inhibition of NF-κB signaling allowed a 3- to 4-fold E₂-mediated induction of ERE₂-luc. This response was both ligand and ER dependent. Repression of ERβ signaling by NF-κB has not previously been reported. Recent evidence suggests that ERβ may function to promote differentiation. The inhibition of ERβ in combination with the antiapoptotic properties of NF-κB may therefore contribute to pathogenesis of GCT.

The mechanisms of granulosa cell tumor (GCT) development may involve the dysregulation of signaling pathways downstream of follicle-stimulating hormone, including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this hypothesis, a genetically engineered mouse model was created to derepress the PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K antagonist gene Pten ( Pten^flox/flox ; Amhr2^cre/+ ). The majority of Pten^flox/flox ; Amhr2^cre/+ mice featured no ovarian anomalies, but occasionally (∼7%) developed aggressive, anaplastic GCT with pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1 was abrogated in Pten^flox/flox ; Amhr2^cre/+ GCT, indicating a mechanism by which GCT cells may increase proliferation and evade apoptosis. To relate these findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT expression were performed on human and equine tumors. Although PTEN loss was not detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has been reported, we tested whether these pathways could synergize in GCT. Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells of a mouse model ( Pten^flox/flox ; Ctnnb1^flox(ex3)/+ ; Amhr2^cre/+ ) resulted in the development of GCT similar to those observed in Pten^flox/flox ; Amhr2^cre/+ mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the ability to spread by seeding into the abdominal cavity. These data indicate a synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the development and progression of GCT and provide the first animal models for metastatic GCT.

The structural determinants of aldosterone binding specificity in the mineralocorticoid receptor (MR) have not been determined. The MR has greatest sequence identity with the better characterized glucocorticoid receptor (GR), which is reflected in their overlapping ligand binding specificities. There must be subtle sequence differences that can account for the MR-specific binding of aldosterone and the shared binding of cortisol. To characterize ligand binding specificity, chimeras were made between the human MR and GR ligand-binding domains (LBDs). Three points were chosen as break points to generate a total of 16 different constructs. These chimeric LBDs were placed in a human GR expression vector containing the GR DNA-binding and N-terminal domains and assayed by co-transfection into CV-1 cells with the mouse mammary tumor virus-luciferase reporter plasmid. Binding of [³H]aldosterone and [³H]dexamethasone was also measured. All of the constructs that are potently activated by aldosterone contain amino acids 804–874 of the MR. The results of the ligand binding experiments using [³H]aldosterone were consistent with the transactivation assay. Cortisol activation of the chimeras was surprisingly complex. Constructs that are activated by cortisol contain either amino acids 804–874 and 932–984 of the MR or amino acids 598–668 and 726–777 of the GR. However, all of the chimeras retained the ability to bind the synthetic glucocorticoid [³H]dexamethasone, and cortisol was able to displace [³H]dexamethasone binding, suggesting that the differential effects of cortisol on transcriptional activation are caused by an effect that occurs downstream of ligand binding. These results identify a subregion of the MR LBD that confers specificity of aldosterone binding, which contrasts with cortisol binding where differential effects between chimeras appear to be mediated by interactions distal to ligand binding.

Development, growth and function of the ovary are controlled by endocrine and paracrine signals. These may also influence the development of ovarian cancer. The aim of this study was to identify the key molecular markers of the unregulated growth and hormone synthesis seen in ovarian tumours, particularly in granulosa cell tumours (GCT). Genes used in this study were chosen on the basis of our understanding of growth and differentiation in the normal ovary. We sought to define the patterns of gene expression in a panel of epithelial and stromal ovarian tumours. Expression was determined by RT-PCR using gene-specific primers for the FSH receptor (FSHR); the FSH early response genes: regulatory subunit of protein kinase A (RII-beta), cyclin D2 (cycD2) and sgk; and late response markers: cyclooxygenase-2 (COX-2) and the LH receptor (LHR). The GCT had high expression of FSHR compared with normal ovaries and the other tumours. cycD2 and RII-beta and COX-2 genes were also highly expressed in the GCT. sgk and LHR expression was lower in all of the tumours than in normal ovaries. Serous cystadenocarcinomas also had an unexpectedly high expression of COX-2. Comparison of the gene expression profiles between each tumour group suggests a molecular phenotype for GCT that is similar to that reported for FSH stimulated pre-ovulatory granulosa cells.