The evolving role of the transfusion practitioner.

Much of the recent work in transfusion practice has shifted to focus on the patient, after efforts over previous decades to ensure the quality and safety of blood products. After the commencement of hemovigilance and transfusion practice improvement programs, the introduction of transfusion practitioners (TP) into health care services and blood centers has continued to increase worldwide. Since this relatively new role was introduced, much work of the TP has focused on patient and staff education, adverse events, transfusion governance, and monitoring of transfusion practices within organizations. The complex nature of the transfusion process makes the TP an integral link in the transfusion chain. Together with hospital transfusion teams and committees, the TP works collaboratively to facilitate the transfusion change management programs and initiatives. Recently, the TP role has evolved to include an emphasis on patient blood management and, to some extent, is shaped by national standards and regulations. These established roles of the TP, together with the ever-changing field of transfusion medicine, provide new opportunities and challenges for a role that is continuing to evolve worldwide.

DHEA metabolites activate estrogen receptors alpha and beta.

Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or ß (ERα or ERß)-regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERß transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERß-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERß-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17ß-estradiol for ERα and ERß binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERßin vitro, modulating estrogen target genes in vivo.

Common data elements for clinical research in Friedreich's ataxia.

To reduce study start-up time, increase data sharing, and assist investigators conducting clinical studies, the National Institute of Neurological Disorders and Stroke embarked on an initiative to create common data elements for neuroscience clinical research. The Common Data Element Team developed general common data elements, which are commonly collected in clinical studies regardless of therapeutic area, such as demographics. In the present project, we applied such approaches to data collection in Friedreich's ataxia (FRDA), a neurological disorder that involves multiple organ systems. To develop FRDA common data elements, FRDA experts formed a working group and subgroups to define elements in the following: ataxia and performance measures; biomarkers; cardiac and other clinical outcomes; and demographics, laboratory tests, and medical history. The basic development process included identification of international experts in FRDA clinical research, meeting by teleconference to develop a draft of standardized common data elements recommendations, vetting of recommendations across the subgroups, and dissemination of recommendations to the research community for public comment. The full recommendations were published online in September 2011 at http://www.commondataelements.ninds.nih.gov/FA.aspx. The subgroups' recommendations are classified as core, supplemental, or exploratory. Template case report forms were created for many of the core tests. The present set of data elements should ideally lead to decreased initiation time for clinical research studies and greater ability to compare and analyze data across studies. Their incorporation into new, ongoing studies will be assessed in an ongoing fashion to define their utility in FRDA.

National Institute of Neurological Disorders and Stroke Common Data Element Project - approach and methods.

BACKGROUND: In neuroscience clinical research studies, much time and effort are devoted to deciding what data to collect and developing data collection forms and data management systems to capture the data. Many investigators receiving funding from National Institute of Neurological Disorders and Stroke (NINDS), the National Institutes of Health (NIH), are required to share their data once their studies are complete, but the multitude of data definitions and formats make it extremely difficult to aggregate data or perform meta-analyses across studies. PURPOSE: In an effort to assist investigators and accelerate data sharing in neuroscience clinical research, the NINDS has embarked upon the Common Data Element (CDE) Project. The data standards developed through the NINDS CDE Project enable clinical investigators to systematically collect data and should facilitate study start-up and data aggregation across the research community. METHODS: The NINDS CDE Team has taken a systematic, iterative approach to develop the critical core and the disease-specific CDEs. The CDE development process provides a mechanism for community involvement and buy-in, offers a structure for decision making, and includes a technical support team. RESULTS: Upon conclusion of the development process, the CDEs and accompanying tools are available on the Project Web site - http://www.commondataelements.ninds.nih.gov/. The Web site currently includes the critical core (aka general) CDEs that are applicable to all clinical research studies regardless of therapeutic area as well as several disease-specific CDEs. Additional disease-specific CDEs will be added to the Web site once they are developed and vetted over the next 12 months. LIMITATIONS: The CDEs will continue to evolve and will improve only if clinical researchers use and offer feedback about their experience with them. Thus, the NINDS program staff strongly encourages its clinical research grantees to use the CDEs and is expanding its efforts to educate the neuroscience research community about the CDEs and to train research teams to incorporate them into their studies. CONCLUSIONS: Version 1.0 of a set of CDEs has been published, but publication is not the end of the development process. All CDEs will be evaluated and revised at least annually to ensure that they reflect current clinical research practices in neuroscience.

Modulation of receptor phosphorylation contributes to activation of peroxisome proliferator activated receptor alpha by dehydroepiandrosterone and other peroxisome proliferators.

Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor alpha (PPARalpha) in vivo but does not ligand-activate PPARalpha in transient transfection experiments. We demonstrate that DHEA regulates PPARalpha action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARalpha and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARalpha mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARalpha mRNA and protein levels as well as increased PPARalpha transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.

The biological actions of dehydroepiandrosterone involves multiple receptors.

Dehydroepiandrosterone has been thought to have physiological functions other than as an androgen precursor. The previous studies performed have demonstrated a number of biological effects in rodents, such as amelioration of disease in diabetic, chemical carcinogenesis, and obesity models. To date, activation of the peroxisome proliferators activated receptor alpha, pregnane X receptor, and estrogen receptor by DHEA and its metabolites have been demonstrated. Several membrane-associated receptors have also been elucidated leading to additional mechanisms by which DHEA may exert its biological effects. This review will provide an overview of the receptor multiplicity involved in the biological activity of this sterol.

Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions.

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.

Transcriptional suppression of cytochrome P450 genes by endogenous and exogenous chemicals.

This article is an invited report of a symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics held at Experimental Biology 2003 in San Diego, California, April 11-15, 2003. Several members of the cytochrome P450 (P450) superfamily are induced after exposure to a variety of chemical signals, and we have gained considerable mechanistic insight into these processes over the past four decades. In addition, the expression of many P450s is suppressed in response to various endogenous and exogenous chemicals; however, relatively little is known about the molecular mechanisms involved. The goal of this symposium was to critically examine our current understanding of molecular mechanisms involved in transcriptional suppression of CYP genes by endogenous and exogenous chemicals. Specific examples were drawn from the following chemical categories: polycyclic and halogenated aromatic hydrocarbon environmental toxicants, inflammatory mediators, the endogenous sterol dehydroepiandrosterone and peroxisome proliferators, and bile acids. Multiple molecular mechanisms are involved in transcriptional suppression, and these processes often involve rather complex cascades of transcription factors and other regulatory proteins. Mechanistic studies of CYP gene suppression can enhance our understanding of how organisms respond to xenobiotics as well as to perturbations in endogenous chemicals involved in maintaining homeostasis.

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.