Lessons from KEEPS: the Kronos Early Estrogen Prevention Study.

The Kronos Early Estrogen Prevention Study (KEEPS) was a randomized, double-blind, placebo-controlled trial designed to determine the effects of hormone treatments (menopausal hormone treatments [MHTs]) on the progression of carotid intima-medial thickness (CIMT) in recently menopausal women. Participants less than 3 years from menopause and without a history of overt cardiovascular disease (CVD), defined as no clinical CVD events and coronary artery calcium < 50 Agatston units, received either oral conjugated equine estrogens (0.45 mg/day) or transdermal 17ß-estradiol (50 µg/day), both with progesterone (200 mg/day for 12 days/month), or placebo pills and patches for 4 years. Although MHT did not decrease the age-related increase in CIMT, KEEPS provided other important insights about MHT effects. Both MHTs versus placebo reduced the severity of menopausal symptoms and maintained bone density, but differed in efficacy regarding mood/anxiety, sleep, sexual function, and deposition of ß-amyloid in the brain. Additionally, genetic variants in enzymes for metabolism and uptake of estrogen affected the efficacy of MHT for some aspects of symptom relief. KEEPS provides important information for use of MHT in clinical practice, including type, dose, and mode of delivery of MHT recently after menopause, and how genetic variants in hormone metabolism may affect MHT efficacy on specific outcomes.

Bioinformatic analysis of benzo-α-pyrene-induced damage to the human placental insulin-like growth factor-1 gene.

INTRODUCTION: Intrauterine growth restriction (IUGR) has been associated with exposure to polyaromatic hydrocarbons (PAHs) which are released in the combustion of oil, fuel, gas, garbage, and tobacco. Pregnant women exposed to PAHs are at risk of the effects of these environmental toxins; for example, benzo-α-pyrene (BαP) is able to enter the blood stream and could contribute to IUGR or other developmental abnormalities via effects on the placental cells. Since IUGR has been associated with decreased cord blood concentrations of immunoreactive insulin-like growth factor 1 (ir-IGF-1) and IUGR has been associated with disordered development and fetal programming, we tested the effects of BαP on human placental trophoblast cells in culture. EXPERIMENTAL: IGF-1 expression and activation was studied using an immortalized human placental trophoblast cell line (HTR-8). The cells were treated with vehicle control or 1 µmol/L BαP, or 5 µmol/L BαP for 12 hours. RNA was extracted and the exons of IGF-1 were amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). The ir-IGF-1 expression levels were compared using gel electrophoresis. The PCR products were sequenced, and levels of mutation were measured with comparative sequence analysis. A computational protein analysis (computer simulation) was performed in order to assess the potential impact of BαP-associated mutation on IGF-1 protein function. RESULTS: The IGF-1 expression decreased considerably in BαP-treated cells relative to untreated controls (P < .05), also in a dose-dependent manner. Comparative sequence analysis indicated that the level of BαP exposure correlated with the percentage of base pair mutations in IGF-1 nucleotide sequences for both treatment groups (P < .05). Shifts were observed in the open reading frame, indicating a possible change in the IGF-1 start codon. Protein folding simulation analysis indicated that the base pair changes induced by BαP weakened IGF-1-IGF binding protein (IGFBP) interaction. CONCLUSIONS: In concordance with the previous findings, exposure of human placental trophoblast cells to BαP exposure results in reduction of IGF-1 expression and base pair mutations. The direct action of BαP on the placenta indicates that it may not be necessary for BαP to access other maternal tissues in order for gene abnormalities to occur. Given that PAHs are known to work through aryl hydrocarbon hydrolase (AHH), these results are likely due to the presence of AHH in HTR cells. Computational modeling of BαP actions on IGF1, substrate-ligand binding, supports the biological premise of this work and underlines the need to determine actual biological effects rather than equating immune to bioactivity of IGF1.

The PBRN initiative: transforming new technologies to improve patient care.

The NIDCR-supported Practice-based Research Network initiative presents dentistry with an unprecedented opportunity by providing a pathway for modifying and advancing the profession. It encourages practitioner participation in the transfer of science into practice for the improvement of patient care. PBRNs vary in infrastructure and design, and sustaining themselves in the long term may involve clinical trial validation by regulatory agencies. This paper discusses the PBRN concept in general and uses the New York University College of Dentistry's Practitioners Engaged in Applied Research and Learning (PEARL) Network as a model to improve patient outcomes. The PEARL Network is structured to ensure generalizability of results, data integrity, and to provide an infrastructure in which scientists can address clinical practitioner research interests. PEARL evaluates new technologies, conducts comparative effectiveness research, participates in multidisciplinary clinical studies, helps evaluate alternative models of healthcare, educates and trains future clinical faculty for academic positions, expands continuing education to include "benchmarking" as a form of continuous feedback to practitioners, adds value to dental schools' educational programs, and collaborates with the oral health care and pharmaceutical industries and medical PBRNs to advance the dental profession and further the integration of dental research and practice into contemporary healthcare (NCT00867997, NCT01268605).

Using basic science to design a clinical trial: baseline characteristics of women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS).

Observational and epidemiological studies suggest that menopausal hormone therapy (MHT) reduces cardiovascular disease (CVD) risk. However, results from prospective trials showed neutral or adverse effects most likely due to differences in participant demographics, such as age, timing of initiation of treatment, and preexisting cardiovascular disease, which reflected in part the lack of basic science information on mechanisms of action of hormones on the vasculature at the time clinical trials were designed. The Kronos Early Estrogen Replacement Study (KEEPS) is a prospective, randomized, controlled trial designed, using findings from basic science studies, to test the hypothesis that MHT when initiated early in menopause reduces progression of atherosclerosis. KEEPS participants are younger, healthier, and within 3 years of menopause thus matching more closely demographics of women in prior observational and epidemiological studies than women in the Women's Health Initiative hormone trials. KEEPS will provide information relevant to the critical timing hypothesis for MHT use in reducing risk for CVD.

Regulation and activation of ezrin protein in endometriosis.

BACKGROUND: Ezrin protein and its activated form phospho-ezrin play a role in cell morphology, motility and adhesiveness. In this study, we hypothesized that these proteins play a role in the pathogenesis of endometriosis by promoting adhesion and invasion of endometrial stromal cells (ESCs) in ectopic sites. METHODS: We compared the expression of ezrin and phospho-ezrin in normal endometrium from women without endometriosis with their expression in eutopic and ectopic endometrial tissues from women with endometriosis, using immunohistochemistry and western blot analysis. Paired eutopic and ectopic endometrial tissue samples from women with endometriosis (n = 13) and normal endometrium from women without endometriosis (n = 12) were collected. Invasive potential of ESCs from each of these samples was compared using Matrigel membrane invasion assay. RESULTS: Eutopic and ectopic endometrial tissues from women with endometriosis have higher ezrin and phospho-ezrin levels as confirmed by immunohistochemistry and western blot analysis (P < 0.05). The Matrigel membrane invasion assay revealed that ectopic ESCs have more invasive characteristics, more protrusions and higher ezrin staining than normal ESCs (P < 0.05). CONCLUSIONS: Ezrin can be a potential marker for endometrial cell invasion and may play a role in the pathogenesis of endometriosis.

Ezrin is a key element in the human vagina.

OBJECTIVE: The vagina is a complex tubular structure that has reproductive, support and barrier functions. These depend on the cytoarchitecture of the vaginal cells, which is controlled by key proteins. Cytoskeletal proteins determine cell polarity and membrane specializations by integrating the actin cytoskeleton with cell membranes. This integration is the domain of cytoskeletal proteins including the MERM protein family (moesin-ezrin-radixin-Merlin). Nothing is known about the cyto-localization of the MERM's in the vaginal epithelium or how it influences the cytoarchitecture of the vaginal epithelium and stroma. DESIGN: Full-thickness human vaginal fornix samples were obtained from 20 normal human specimens obtained at surgery for pelvic relaxation. Light- and electron microscopical immunohistochemistry (IHC) were used to identify and study activation and cellular localization of immuno-reactive-ezrin (ir-ezrin), a prototypical MERM. RESULTS: Ir-ezrin was identified in the stratified squamous vaginal epithelium and connective tissue (fibroblasts, blood vessels and leucocytes). "H" scoring indicated that ir-ezrin staining is denser in the vaginal epithelium than in other layers, that the ir-ezrin staining was associated with increased keratinization and with the size of the tight junctions (p<0.01). Both the amounts and localization of ir-ezrin were associated with high levels of estrogen, identified by the menstrual history and keratinization of the superficial vaginal epithelium. The density of stromal ir-ezrin was increased in the presence of dense epithelial keratinization. Immuno-reactive-ezrin staining was most pronounced near the cell membranes of both keratinized and non-keratinized epithelium, indicating that ezrin activation (unfolding and movement to the membrane) had occurred. Ultra-structural examination of the epithelium showed intra-cellular ir-ezrin to be localized to junctional complexes that have been associated with decreased mucosal penetration by microorganisms. Ir-ezrin was widely distributed throughout stromal fibro-muscular cell, vessels and immunocytes. CONCLUSIONS: MERM's, represented by ezrin, are widely present in the vaginal wall. This has implications for the strength and resilience of this tubular structure and may be the case in other internal genital tissues. Ezrin's localization and association with cell specializations indicate that in the vagina, as in other tissues, ezrin likely modulates vaginal cell-cell interactions including the changing vaginal cellular interface with the external environment, the regulation of the elasticity of the vagina, and the regulation of microbial and chemical traffic that determine the pH and microbial environment of the vagina. In other work we have shown that ezrin expression is induced by estradiol. The increase of ir-ezrin staining during the appearance of keratinization and maturation of the vaginal cytology indicates that estrogen may regulate vaginal ezrin and thereby the properties of the vaginal wall and epithelium.

Protein misfolding and misprocessing in complex disease.

Scientists from over 20 major research centers recently convened to discuss advances and new discoveries in "Protein MisFolding and MisProcessing in Disease." Understanding protein mechanisms the underlying etiology of complex diseases lies in analyzing the associated biochemical mechanisms, which include folding patterns, processing patterns, chaperone regulators, stress pathways, and signal transduction.

Circulation DNA: biological implications for cancer metastasis and immunology.

Circulation cell free DNA (cf-DNA) is of considerable interest to oncology researchers seeking to isolate specific cancer markers. Here, we focus on the origin and biological implications of cf-DNA, exploring its potential roles in cancer biology and medicine. We hypothesize that cf-DNA is primarily released by living cancer cells in addition to apoptotic or necrotic cancer cells for three reasons: (1) following radiotherapy, cf-DNA quantities are significantly reduced in a high percentage of patients although radiation-induced massive apoptosis is expected; (2) cancer cell DNA concentration in cultured supernatants increases with cell proliferation when few apoptotic or necrotic cells are present; and (3) DNA concentration increases in normal lymphocyte cultures following stimulation with phytohemagglutinin, lipopolysaccharide or antigen. Our hypotheses have major biological implications in cancer biology. First, cancer cf-DNA may transform normal cells and form adjacent or remote metastases or second primary cancer. In this context, we also have raised an alarming advice that the cancer may be potentially infectious. Secondly, if a normal cf-DNA contains cytokine sequence, it may behave like an intrinsic DNA vaccine, producing therapeutic cytokine. If normal cf-DNA contains a sequence of a non-mutated oncogene or tumor suppressor gene, homologous recombination with the cancer genome may occur leading to knock out mutated oncogene or tumor suppressor gene that could thus elicit a spontaneous remission of cancer.

KEEPS: The Kronos Early Estrogen Prevention Study.

Observational studies have indicated that hormone therapy given at or after menopause is linked to substantial reduction in cardiovascular disease and its risk factors. Recent findings from the Women's Health Initiative (WHI) clinical trial, however, indicate that combined estrogen plus progestin hormone therapy, as well as estrogen-alone hormone therapy (given to women without a uterus), is ineffective in preventing the new onset of cardiac events in previously healthy late menopausal women. Further, the secondary prevention trial, the Heart and Estrogen/progestin Replacement Study (HERS), also failed to demonstrate any benefit of initiation of hormone therapy in women with established coronary heart disease. In light of these results, a hypothesis has arisen that early initiation of hormone therapy, in women who are at the inception of their menopause, will delay the onset of subclinical cardiovascular disease in women. The rationale that earlier intervention than that performed in the WHI and HERS trials will provide cardiovascular benefit to women is the driving force behind the Kronos Early Estrogen Prevention Study, or KEEPS. KEEPS is a multicenter, 5-year clinical trial that will evaluate the effectiveness of 0.45 mg of conjugated equine estrogens, 50 microg weekly transdermal estradiol (both in combination with cyclic oral, micronized progesterone, 200 mg for 12 days each month), and placebo in preventing progression of carotid intimal medial thickness and the accrual of coronary calcium in women aged 42-58 years who are within 36 months of their final menstrual period. A total of 720 women are planned to be enrolled in 2005, with an anticipated close-out of the trial in 2010. This overview summarizes the recruitment and methodology of the KEEPS trial.

The influence of environmental adaptation on bacterial genome structure.

AIMS: Researchers have long been puzzled by the diversity of life. Now that the complete genomic sequence of many organisms has been determined, it is possible to evaluate the impact of organismal variation on sequence structure or vice versa. The aim of this investigation was to explore genomic changes mandated by organismal adaptation to its ecological niches. METHODS AND RESULTS: Coding sequences from three phylogenetically related bacterial species namely Mycoplasma genitalium, M. pneumoniae and Ureaplasma urealyticum were subject to in depth sequence analyses. M. genitalium and M. pneumoniae both belong to the genus Mycoplasma while U. urealyticum is a member of the genus Ureaplasma. However, M. genitalium and U. urealyticum are urogenital pathogens while M. pneumoniae is a respiratory pathogen. Complete transcriptomes were downloaded from NCBI for each species, and were subject to in silico investigation using in-house software, and public sequence analysis tools. Clear similarities in transcriptome structure were identified among the functionally similar species M. genitalium and U. urealyticum while no such relationship was identified among the phylogenetically related species M. genitalium and M. pneumoniae. CONCLUSIONS: It is plausible to conclude that, in these bacterial species, environmental stimuli might be more influential in shaping sequence signatures than phylogenetic relationships. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that molecular signatures within the transcriptomes of the species examined are likely to be a product of evolutionary adaptation to diverse environmental ecological stimuli, and not a result of common phylogeny.

Diagnostic microarray: an array of possibilities for parasitology.

Parasites affect a majority of the world's population. Despite this fact, dreams of developing vaccines remain far off. Scientists have long studied gene expression as a hallmark of gene activities reflecting current cell conditions. Analyzing differentially expressed genes is a major initiative, and most labs recoil at the amount of time and high costs required obtaining results. By employing microarrays, researchers can decrease their reliance upon time consuming techniques; consequently, microarray is beginning to dominate other molecular diagnostic technologies. Moreover, the ability of microarrays to monitor simultaneous gene expression of thousands of genes and to produce broad arrays of data has the potential to shift the resources of the scientists from data gathering to analyzing data that are already available. As microarray technology improves and its cost decreases, the role of ability to "see" the molecular biology pathways involved in parasite host relationships will place this technology at the forefront of parasite research.

New approaches to the understanding of tamoxifen action and resistance.

Tamoxifen (TAM) provides an effective agent for treatment of hormone-dependent breast cancer but resistance uniformly ensues upon continued use. Additional studies are required to define more precisely the mechanisms involved in development of resistance. We conducted systematic experimental and clinical studies based on the hypothesis that tumors exposed to TAM long-term may develop resistance by becoming hypersensitive to its estrogenic effects. These investigations uncovered new features of the TAM resistance (TR) phenomenon and identified possible means for its prevention and/or elimination. Initially we confirmed that TR may be divided into two subtypes, primary and acquired resistance, and that these differ by certain important characteristics including the level of the possible involvement of adaptive and genetic components. Then we distinguished at least three consequent stages of this phenomenon: stage I when TAM behaves as an antiestrogen, stage II with development of increased sensitivity to the agonistic (pro-estrogenic) properties of TAM and stage III with an adaptive increase in sensitivity to estradiol (E(2)). During this evolutionary process, as shown in vitro, MAP kinase (MAPK) and aromatase activities increase. The time frame of the increase in MAPK activity as a rule outpaces the increase in aromatase activity during the course of the development of TR. This may occur as a response to estrogen deprivation or interruption of the process of estrogen signaling and can be one of the promoting factors of increased aromatase activation. On the other hand, the chronology of these events indicates that changes in the MAPK cascade can be more important for the early steps of the development and maintenance of the TR state. Changes in local estrogen production/sensitivity to E(2) are perhaps essential for the later steps of this phenomenon. We have explored the use of a growth factor-blocking agent to abrogate the adaptive changes in sensitivity. Farnesylthiosalicylic acid (FTS), an inhibitor of GTP-Ras binding to its membrane acceptor site, reduces the increase in the number of MCF-7 cells induced by long-term TAM treatment. It also decreases MAPK activity in TAM-treated MCF-7 cells and in established TR cell lines. Alone or in combination with letrozole (presumably, through the influence on MAPK pathway) FTS exerts moderate inhibitory effects on aromatase activity in estrogen-deprived or estrogen-exposed MCF-7 cells. Taken together, our observations suggest that FTS is a 'candidate drug' for the treatment of TR. Both the adaptive and genetic types of resistance may be amenable to this approach. Our studies underline the possible importance of starting the treatment/prevention of TR early on. From our clinical studies using immunohistochemistry, there is a rather strong rationale to include as a predisposing factor in the development of TR the increase in MAPK and aromatase activities in human primary breast tumors. In summary, data obtained during the course of this project may be considered as evidence supporting the principle that processes resulting in responses to TAM as an agonist and the development of estrogen hypersensitivity of breast cancer cells could potentially be mechanistically linked.

Estradiol affects axo-somatic contacts of neuroendocrine cells in the arcuate nucleus of adult rats.

It has been shown that gonadal steroids have the capacity to induce synaptic plasticity in certain areas of the nervous system. Previously we have demonstrated that due to the effect of estradiol there is a transient decrease in the number of GABAergic axo-somatic synapses in the arcuate nucleus. By using systemic application of the tracer Fluorogold we retrogradely labeled a subpopulation of arcuate neurons that project to the median eminence. We than applied the disector method for synapse quantification and found that these "hypophysiotropic neurons" receive less axo-somatic inputs. We found that 17beta-estradiol induced a decrease in the numerical density of axo-somatic contacts of these retrogradely-labeled neoroendocrine cells. Our data support the hypothesis that the hormonally driven morphological synaptic plasticity is neuron specific within the arcuate nucleus and plays a decisive role in the regulation of anterior pituitary.

Estrogen, synaptic plasticity and hypothalamic reproductive aging.

Unlike primates who undergo ovarian failure and loss of sex steroids at the end of reproduction, aging rodents undergo constant vaginal estrus followed by constant diestrus and finally anestrus, which indicates the absence of responsive ovarian follicles. The latter state is analogous to menopause in women. The timing of the appearance of constant estrus is determined by many factors including estrogen exposure in the brain during development and the number of times that the animal gets pregnant. The chief site of this reproductive aging in rat brains is the arcuate nucleus of the hypothalamus. The transition from normal cycles to constant estrus parallels the females' gradually decreased ability to respond to administered estradiol with a cycle of inhibition followed by disinhibition of gonadotrophin-releasing hormone. Evidence has accumulated indicating this to be due to a loss of the rat's ability to respond to markedly elevated estradiol with the usual arcuate nucleus neuro-glial plasticity that supports the estrogen-induced gonadotrophin surge (EIGS). Just as male rats are not capable of an EIGS, aged females loose this ability through repeated EIGS. Experiments indicate that in male rats the hypothalamic synaptology that develops as a result of exposure to testicular androgens in the perinatal period (brain sexual differentiation) is a result of conversion of testosterone from the testes to estrogen in the brain and is therefore due to early estrogen exposure. Aging females appear to reach a synaptology similar to males and constant estrus as a result of repeated exposure to ovarian estrogens during their reproductive careers. The relative role of aging and hormonal factors remains unclear. Morphological evidence is presented that indicates the above effects of estrogen involve changes in hypothalamic arcuate nucleus neurons and glia, including changes in the organization of perikaryal membranes as well as arcuate nucleus synaptology and the load of peroxidase in the astroglia. A possible role for free radicals (reactive oxygen species) in hypothalamic reproductive aging has been proposed. Such a mechanism is supported by evidence that the anti-oxidant vitamin E delays the onset of constant estrus and the accumulation of glial peroxidase in aging female rats. However, since the synaptology and peroxidase load in constant estrus females is independent of the age at which the constant estrus occurs, it appears that the role of (repeated) estradiol exposure is more deterministic of hypothalamic failure than is aging, per se.