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Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The single-nucleotide polymorphism (SNP) rs2887571 is predicted from genome-wide association studies (GWASs) to associate with osteoporosis but has had an unknown mechanism. Analysis of osteoblasts from 110 different individuals who underwent joint replacement revealed that the genotype of rs2887571 correlates with WNT5B expression. Analysis of our ChIP-sequencing data revealed that SNP rs2887571 overlaps with an estrogen receptor alpha (ERα) binding site. Here we show that 17β-estradiol (E2) suppresses WNT5B expression and further demonstrate the mechanism of ERα binding at the enhancer containing rs2887571 to suppress WNT5B expression differentially in each genotype. ERα interacts with NFATc1, which is predicted to bind directly at rs2887571. CRISPR-Cas9 and ChIP-qPCR experiments confirm differential regulation of WNT5B between each allele. Homozygous GG has a higher binding affinity for ERα than homozygous AA and results in greater suppression of WNT5B expression. Functionally, WNT5B represses alkaline phosphatase expression and activity, decreasing osteoblast differentiation and mineralization. Furthermore, WNT5B increases interleukin-6 expression and suppresses E2-induced expression of alkaline phosphatase during osteoblast differentiation. We show that WNT5B suppresses the differentiation of osteoblasts via receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), which activates DVL2/3/RAC1/CDC42/JNK/SIN3A signaling and inhibits β-catenin activity. Together, our data provide mechanistic insight into how ERα and NFATc1 regulate the non-coding SNP rs2887571, as well as the function of WNT5B on osteoblasts, which could provide alternative therapeutic targets for osteoporosis.

Progestins, synthetic compounds designed to mimic the activity of natural progesterone (P4), are used globally in menopausal hormone therapy. Although the older progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) have been implicated in increased breast cancer risk, little is known regarding newer progestins, and no significant risk has been associated with P4. Considering that breast cancer is the leading cause of mortality in women, establishing which progestins increase breast cancer incidence and elucidating the underlying mechanisms is a global priority. We showed for the first time that the newer-generation progestin drospirenone (DRSP) is the least potent progestin in terms of proliferation of the estrogen-responsive MCF-7 BUS breast cancer cell line, while NET and P4 have similar potencies to estradiol (E2), the known driver of breast cancer cell proliferation. Notably, MPA, the progestin most frequently associated with increased breast cancer risk, was significantly more potent than E2. While all the progestogens enhanced the anchorage-independent growth of the MCF-7 BUS cell line, MPA promoted a greater number of colonies than P4, NET or DRSP. None of the progestogens inhibited E2-induced proliferation and anchorage-independent growth. We also showed that under non-estrogenic conditions, MPA and NET, unlike P4 and DRSP, increased the expression of the estrogen receptor (ER) target gene, cathepsin D, via a mechanism requiring the co-recruitment of ERα and the progesterone receptor (PR) to the promoter region. In contrast, all progestogens promoted the association of the PR and ERα on the promoter of the PR target gene, MYC, thereby increasing its expression under non-estrogenic and estrogenic conditions. These results suggest that progestins differentially regulate the way the PR and ER converge to modulate the expression of PR and ER-regulated genes. Our novel findings indicating similarities and differences between P4 and the progestins, emphasize the importance of comparatively investigating effects of individual progestins rather than grouping them as a class. Further studies are required to underpin the clinical relevance of PR/ERα crosstalk in response to different progestins in both normal and malignant breast tissue, to either confirm or refute their suitability in combination therapy for ER-positive breast cancer.

Objectives We described the epidemiology and healthcare exposures during a measles outbreak in London and identified factors associated with isolation on arrival to healthcare premises. Study design We conducted a cohort study including all confirmed measles cases in London residents from February 1, 2016, to June 30, 2016, and semistructured interviews with two infection prevention and control teams (IPCTs). Methods We described the outbreak and conducted a multilevel mixed-effects analysis to assess the relationship between sociodemographic and clinical factors and isolation on arrival to healthcare premises. We summarised the interviews. Results There were 182 cases, mostly aged 17–35 years (46%; 84). Excluding cases younger than one year, 76% (92/120) were unvaccinated, including two healthcare workers. The majority presented with rash (97%; 174), and 42% (70/166) required hospitalisation. Of the recorded cases, 93% of cases (164/178) had visited a healthcare setting during their infectious period (median number of visits = 2). In 33% (59/178) of the visits, the case was isolated on arrival; when not isolated, four healthcare exposures resulted in further transmission. Presenting to the hospital as opposed to a general practitioner (GP) was associated with higher odds of isolation (odds ratio = 2.23, 95% confidence interval = 1.1–4.4) when adjusted for age, gender and presenting with a cough. The IPCT identified measles training using standardised risk assessments by triage nurses in accident and emergency and intelligence regarding measles activity in the community as positive measures to prevent healthcare exposures. Conclusions We recommend opportunistic immunisation of unvaccinated young adults by GPs and that occupational health departments ensure their staff are protected against measles. Raising measles awareness in healthcare settings via training or regular sharing of current measles surveillance activity from public health to the IPCT and GP may improve triage and isolation of cases on arrival to healthcare premises.

Background Hospital professionals must attend to patients’ satisfaction with care. Along with technical quality of care, patients’ personal characteristics may affect that satisfaction, but standard demographics research often overlooks cultural links. Methods We, therefore, asked 58 San Antonio, Texas, inpatients their satisfaction with care and examined responses for attitudes related to ethnic—Mexican-American (MA), Euro-American (EA), or African-American (AA)—and gender cultures. Results Many attitudes occurred widely. Most respondents expected doctors to attend them faithfully, inform them honestly, and pursue their needs and wishes singularly. Most also trusted doctors, and expressed satisfaction with doctors’ generally exemplary character and service ethic. But most respondents also feared hospital treatments, and some expressed dissatisfaction that doctors had inadequately informed them or ignored their wishes. Only rare attitudes distinguished particular ethnic-gender groups. Unlike other groups few EA or AA men expressed dissatisfactions. But some MA and EA women said hospitals use too many caregivers or coordinate care poorly. Furthermore, most AA women expressed no explicit trust in doctors, and most EA women expressed actual distrust of doctors, often doubting their technical competence or altruism. Conclusions These findings suggest a novel perspective: a unique inpatient culture, largely unaffected by ethnic group or gender. Patients interpret their hospital experience through that culture. Hospital professionals might respond with both universal measures (addressing patients’ fears, dissatisfactions, and distrust) and targeted ones (explicitly asking EA and AA men about dissatisfactions, and AA and EA women about distrust). Such culturally grounded measures may help maintain or increase inpatients’ satisfaction.

Following an extensive contact tracing exercise at a school in a London borough with one of highest tuberculosis (TB) rates in England, we estimated the background prevalence of latent TB infection to be significantly less than the widely accepted 10%. We screened 271 pupils aged 14–15 years in two groups: 96 pupils in group 1 had significant exposure (>8 h/week in the same room) to a case of infectious TB and 175 in group 2 who had minimal exposure. In group 1, 26% were diagnosed with latent or active TB, compared to 6.3% in group 2. Risk factors for TB infection (e.g. previous exposure or link to high-prevalence communities) were analysed using a cohort study design. In the univariable analysis only being in contact group 1 was statistically significantly associated with being a case (OR 5.25, 95%,P< 0.001). In the multivariable model contact group 1 remained significantly associated with being a case (adjusted OR 4.40,P= 0.001). We concluded that the 6.3% yield of TB infection in contact group 2 is either similar to or higher than the background prevalence rate of latent TB infection (LTBI) in this high TB prevalence London borough. Other parts of England with lower TB prevalence are likely to have even lower LTBI rates.

Summary Background Rheumatic heart disease (RHD) is a major public health problem in low– and middle–income countries (LMICs), with a paucity of high–quality trial data to improve patient outcomes. Investigators felt that involvement in a recent large, observational RHD study impacted positively on their practice, but this was poorly defined. Aim The purpose of this study was to document the experience of investigators and research team members from LMICs who participated in a prospective, multi–centre study, the global Rheumatic Heart Disease Registry (REMEDY), conducted in 25 centres in 14 countries from 2010 to 2012. Methods We conducted an online survey of site personnel to identify and quantify their experiences. Telephone interviews were conducted with a subset of respondents to gather additional qualitative data. We asked about their experiences, positive and negative, and about any changes in RHD management practices resulting from their participation in REMEDY as a registry site. Results The majority of respondents in both the survey and telephone interviews indicated that participation as a registry site improved their management of RHD patients. Administrative changes included increased attention to follow–up appointments and details in patient records. Clinical changes included increased use of penicillin prophylaxis, and more frequent INR monitoring and contraceptive counselling. Conclusions Our study demonstrates that participation in clinical research on RHD can have a positive impact on patient management. Furthermore, REMEDY has led to increased patient awareness and improved healthcare workers’ knowledge and efficiency in caring for RHD patients.

Progestins used in endocrine therapies bind to multiple steroid receptors and are associated with several side-effects. It is thus important to understand the relationship between steroid receptor cross-reactivity and the side-effect profile of progestins. In cell lines that express negligible levels of steroid receptors, we report for the first time the binding affinities, potencies and efficacies of selected progestins from different generations determined in parallel. We show that the progestins bind to the androgen receptor (AR) with similar affinities to each other and progesterone, while none bind estrogen receptor (ER)-β, and only norethisterone acetate, levonorgestrel and gestodene bind ERα. Comparative dose-response analysis revealed that progestins from the first three generations display similar androgenic activity to the natural androgen dihydrotestosterone for transactivation, while norethisterone acetate, levonorgestrel and gestodene are ERα agonists. We show for the first time that the anti-androgenic properties of progesterone and drospirenone are similar to the well-known AR antagonist hydroxyflutamide, while nomegestrol acetate is more potent and nestorone less potent than both hydroxyflutamide and progesterone. Moreover, we are the first to report that the older progestins, unlike progesterone and the fourth generation progestins, are efficacious ERα agonists for transrepression, while the selected progestins from the second and third generation are efficacious AR agonists for transrepression. Considering the progestin potencies and their reported free serum concentrations relative to dihydrotestosterone and estradiol, our results suggest that the progestins are likely to exert AR-, but not ERα- or ERβ-mediated effects in vivo.

sectitleSETTING/titleThe incidence of multidrug-resistant tuberculosis (MDR-TB) is routinely reported by the Public Health England, UK, but prevalence better represents burden./secsectitleOBJECTIVE/titleTo estimate MDR-TB prevalence, and identify the factors associated with acquired resistance and unsuccessful outcomes in people managed by the health services./secsectitleDESIGN/titleWe included notified MDR-TB cases prevalent between 2010 and 2014. Multivariable logistic regression was used to identify the factors associated with acquisition of resistance and unsuccessful outcomes. The social risk factors (SRFs) recorded were alcohol, drug misuse, homelessness and incarceration./secsectitleRESULTS/titleBetween 2010 and 2014, there were 2.3-3.1 times more prevalent than incident cases each year, with an increasing prevalence-to-incidence ratio over time; 86% of prevalent cases were foreign-born, and 15% had an SRF. Overall, 11% of MDR-TB cases acquired resistance, including 18% of those with SRFs and 22% of UK-born MDR-TB cases acquired resistance. Half of the cases completed treatment by 24 months; those with SRFs, extensive drug resistance or acquired resistance were less likely to complete treatment./secsectitleDISCUSSION/titleThe number of prevalent cases is higher than incident cases, and increases over time, so a focus on prevalent cases enables better planning for services to support patients. We recommend that additional support be provided for those at risk of acquiring resistance, including those with SRFs./sec.

Background/objectives Ideally, doctors ask each patient's current views about involvement in decision-making, but inquiries prove inconclusive with some inpatients. Doctors may then need indirect indicators of those views. We, therefore, explored ethnic group and sex as cultural indicators of patients' current preferences and perceptions about such involvement. Methods In open-response interviews, we asked those preferences and perceptions of 26 Mexican American (MA), 18 Euro-American (EA), and 14 African American (AA) adult inpatients. We content-analyzed responses blindly to identify themes and linked those themes to ethnic group and sex. Results Only sex indicated patients' current preferences. Regardless of ethnic group, most men preferred decision-making by the doctor (with or without the patient); most women, decision-making by the patient (with or without the doctor). But both ethnic group and sex together indicated patients' current perceptions. Specifically, each ethnic group as a whole most often perceived decision-making by the doctor alone and the patient alone on separate occasions, but the sexes within ethnic groups differed. For MAs roughly equal numbers of men and women perceived such decision making, for EAs more men than women did so, and for AAs more women than men did so. In addition, no EA men but some EA women perceived decision-making by the doctor alone, and some MA men and women-but no EAs or AAs-perceived decision-making by the patient alone. Primarily ethnic group indicated matches between current preferences and perceptions: Most EAs had matches; most MAs and AAs did not. Conclusions Whenever direct inquiries fail, ethnic group and sex may indicate adult inpatients' current preferences and perceptions about involvement in decision-making. Yet matching those preferences and perceptions, especially for minority patients, remains difficult.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.

As in other semi-arid savannah systems of the world characterised by highly mobile and/or migratory ungulates, Botswana's rangelands are experiencing increased fragmentation due to expanding human activities and increasing human wildlife conflict. Climate change scenarios show Botswana becoming hotter and drier with mega droughts, heat waves and more intense and spatially confined rainfall events. The Botswana Government has reacted by providing artificial water points (AWPs) in the Protected Areas and surrounding Wildlife Management Areas (WMAs), in part to compensate for the lack of access to historical sources due to fences and human expansion. Blanket provision of AWPs will disrupt the existing mobility and migratory strategies of the key ungulates that is basic to their survival and their ability to adapt to climate change. Botswana's burgeoning elephant population has already effectively re-connected the drier Kalahari System to the Northern System by breaching fences in the region. The key recommendations from the past are used to reinforce the need for ecosystem management for resilience at the landscape level via migratory corridors through shared landscapes, made possible by a renewed focus on Community Based Natural Resource Management and Payments for Ecosystem Services. The events that have occurred over the last 50 years are used to illustrate the dangers of managing at the wrong 'localised' spatial and temporal scale and failing to address the key factors of mobility and inequity that characterise the ecological and socio-economic systems, respectively.

The Scientific Foundation ‘Asociación Española Contra el Cáncer’ (AECC, Stable Coordinated Group, Hereditary Cancer); the BBVA Foundation; the Eugenio Rodríguez Pascual Foundation grant 2012; Generalitat de Catalunya AGAUR SGR 2012 grants 283, 290 and 312, and SGR 2014 grants 364, 530, and 535; Spanish Ministry of Health ISCIII FIS grants PI10/00057, PI10/00222, PI10/01422, PI12/01528, PI13/00132, and PI14/00336. ISCIII RTICC grants RD06/0020/1051, RD12/0036/0007, RD12/0036/0008 and RD12/0036/0063; Spanish Ministry of Science and Innovation, ‘Fondo Europeo de Desarrollo Regional (FEDER), una manera de hacer Europa’, MINECO grants SAF2010-20203 and SAF2013-46196; and the Telemaraton 2014 ‘Todos Somos Raros, Todos Somos Únicos’ grant P35 (...), Mateo, F.; Arenas, E. J.; Aguilar, H.; Serra-Musach, J.; de Garibay, G. Ruiz; Boni, J.; Maicas, M.; Du, S.; Iorio, F.; Herranz-Ors, C.; Islam, A.; Prado, X.; Llorente, A.; Petit, A.; Vidal, A.; Catala, I.; Soler, T.; Venturas, G.; Rojo-Sebastian, A.; Serra, H.; Cuadras, D.; Blanco, I.; Lozano, J.; Canals, F.; Sieuwerts, A. M.; de Weerd, V.; Look, M. P.; Puertas, S.; Garcia, N.; Perkins, A. S.; Bonifaci, N.; Skowron, M.; Gomez-Baldo, L.; Hernandez, V.; Martinez-Aranda, A.; Martinez-Iniesta, M.; Serrat, X.; Ceron, J.; Brunet, J.; Barretina, M. P.; Gil, M.; Falo, C.; Fernandez, A.; Morilla, I.; Pernas, S.; Pla, M. J.; Andreu, X.; Segui, M. A.; Ballester, R.; Castella, E.; Nellist, M.; Morales, S.; Valls, J.; Velasco, A.; Matias-Guiu, X.; Figueras, A.; Sanchez-Mut, J. V.; Sanchez-Cespedes, M.; Cordero, A.; Gomez-Miragaya, J.; Palomero, L.; Gomez, A.; Gajewski, T. F.; Cohen, E. E. W.; Jesiotr, M.; Bodnar, L.; Quintela-Fandino, M.; Lopez-Bigas, N.; Valdes-Mas, R.; Puente, X. S.; Vinals, F.; Casanovas, O.; Graupera, M.; Hernandez-Losa, J.; Ramon y Cajal, S.; Garcia-Alonso, L.; Saez-Rodriguez, J.; Esteller, M.; Sierra, A.; Martin-Martin, N.; Matheu, A.; Carracedo, A.; Gonzalez-Suarez, E.; Nanjundan, M.; Cortes, J.; Lazaro, C.; Odero, M. D.; Martens, J. W. M.; Moreno-Bueno, G.; Barcellos-Hoff, M. H.; Villanueva, A.; Gomis, R. R.; Pujana, M. A.

Conventional hormone therapy (HT) containing estrogens such as ethinylestradiol (EE) have been associated with an increased risk of breast cancer and cardiovascular disease resulting in women seeking safer alternatives that are claimed to have fewer health risks. One such alternative gaining popularity, is custom-compounded bioidentical (b)HT formulations containing bioidentical estradiol (bE2) and estriol (bE3). However, the preparation of these custom-compounded estrogens is not regulated, and depending on the route of synthesis, steroid mixtures with differing activities may be produced. Thus, an investigation into the activities of estrogens prepared by custom-compounded pharmacies is warranted. The aim of this study was therefore to directly compare the pharmacological properties of bE2 and bE3 of unknown purity relative to commercially available, pure E2, E3 and estrone (E1) standards as well as synthetic EE used in conventional HT via the human estrogen receptor (ER)-α and -β. We determined precise equilibrium dissociation constants (Kd or Ki values) and showed that bE2 and bE3 display similar binding affinities to the E2 and E3 standards, while EE had a higher affinity for ERα, and E1 a lower affinity for ERβ. Furthermore, all the estrogens display similar agonist efficacies, but not potencies, for transactivation on a minimal ERE-containing promoter via the individual ER subtypes. Although E2 and E3 were equally efficacious and potent on the endogenous ERE-containing pS2 promoter in the MCF-7 BUS breast cancer cell line co-expressing ERα and ERβ, E1 was less efficacious and potent than E2. This study is the first to demonstrate that the bioidentical estrogens, commercially available estrogen standards and synthetic EE are full agonists for transrepression on both minimal and endogenous NFκB-containing promoters. Moreover, we showed that these estrogens all increase proliferation and anchorage-independent growth of MCF-7 BUS cells to a similar extent, suggesting that custom-compounded bHT may in fact not be a safer alternative to conventional HT. Furthermore, our results showing that E3 and E1 are not weak estrogens, and that E3 does not antagonize the activity of E2, suggest that the rationale behind the use of E3 and E1 in custom-compounded bHT formulations should be readdressed. Taken together, the results indicating that there is mostly no difference between the custom-compounded bioidentical estrogens, commercially available estrogen standards and synthetic EE, at concentrations reflecting serum levels in women using estrogen-containing HT, suggest that there is no clear advantage in choosing bHT above conventional HT.

The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1-EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention.

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). Under castrate conditions, DHT is produced from the metabolism of the adrenal androgen precursors, DHEA and androstenedione. Recent studies have shown that the adrenal steroid 11β-hydroxyandrostenedione (11OHA4) serves as the precursor to the androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). In this study we comprehensively assess the androgenic activity of 11KT and 11KDHT. This is the first study, to our knowledge, to show that 11KT and 11KDHT, like T and DHT, are potent and efficacious agonists of the human androgen receptor (AR) and induced both the expression of representative AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines, LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells, while in vitro conversion assays showed that 11KT and 11KDHT are metabolized at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT, respectively. Our findings show that 11KT and 11KDHT are bona fide androgens capable of inducing androgen-dependant gene expression and cell growth, and that these steroids have the potential to remain active longer than T and DHT due to the decreased rate at which they are metabolised. Collectively, our data demonstrates that 11KT and 11KDHT likely play a vital, but overlooked, role in the development and progression of CRPC.

Progestins used in contraception and hormone replacement therapy are synthetic compounds designed to mimic the actions of the natural hormone progesterone and are classed into four consecutive generations. The biological actions of progestins are primarily determined by their interactions with steroid receptors, and factors such as metabolism, pharmacokinetics, bioavailability and the regulation of endogenous steroid hormone biosynthesis are often overlooked. Although some studies have investigated the effects of select progestins on a few steroidogenic enzymes, studies comparing the effects of progestins from different generations are lacking. This study therefore explored the putative modulatory effects of progestins on de novo steroid synthesis in the adrenal by comparing the effects of select progestins from the respective generations, on endogenous steroid hormone production by the H295R human adrenocortical carcinoma cell line. Ultra-performance liquid chromatography/tandem mass spectrometry analysis showed that the fourth-generation progestins, nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP), unlike the progestins selected from the first three generations, modulate the biosynthesis of several endogenous steroids. Subsequent assays performed in COS-1 cells expressing human 3βHSD2, suggest that these progestins modulate the biosynthesis of steroid hormones by inhibiting the activity of 3βHSD2. The Ki values determined for the inhibition of human 3βHSD2 by NES (9.5 ± 0.96 nM), NoMAC (29 ± 7.1 nM) and DRSP (232 ± 38 nM) were within the reported concentration ranges for the contraceptive use of these progestins in vivo. Taken together, our results suggest that newer, fourth-generation progestins may exert both positive and negative physiological effects via the modulation of endogenous steroid hormone biosynthesis.

A series of new water soluble Ru(III) pyrazole complexes mer-[RuCl3(DMSO-S)(pyz)2] 1, mer-[RuCl3(DMSO-S)(DMSO-O)(pyz)] 2, mer-[RuCl3(bpy)(dmpyz)] 3, and mer-[RuCl3(DMSO-S)(dmpyz)2] 4 (pyz = pyrazole; dmpyz = 3,5-dimethylpyrazole, bpy = 2,2’-bipyridine) have been synthesized and characterized by use of a combination of spectroscopy (IR and UV-visible), X-ray diffraction, and cyclic voltammetry. The molecular X-ray structure of all reported compounds (1–4) revealed distorted octahedral coordination around ruthenium. The cytotoxicity assay on human breast cancer cells (MCF7) demonstrated that compounds 1 and 4 affect cell viability, whereas compounds 2 and 3 do not show appreciable activity. The IC50 values for 1 and 4 lie within the range of 71–32 μM in MCF7 cells.

Background Because beliefs about the soul after death affect the dying experience, patients and survivors may want to discuss those beliefs with their healthcare provider; however, almost no medical research describes such beliefs, leaving healthcare professionals ill prepared to respond. This exploratory study begins the descriptive process. Methods Assuming that culture is key, we asked older adult Mexican American (MA), European American (EA), and African American (AA) inpatients their beliefs about whether the soul lives on after physical death; if so, where; and what the "afterlife" is like. Results Some beliefs varied little across the sample. For example, most participants said that the soul lives on after physical death, leaves the body immediately at death, and eventually reaches heaven. Many participants also said death ends physical suffering; however, other beliefs varied distinctly by ethnic group or sex. More AAs than MAs or EAs said that they believed that the soul after physical death exists in the world (57% vs 35% and 33%) or interacts with the living (43% vs 31% and 28%). Furthermore, in every ethnic group more women than men said they believed that the soul exists in the world (42% vs 29% for MAs, 45% vs 14% for EAs, and 71% vs 43% for AAs). Conclusions As death nears, patients or survivors may want to discuss beliefs about the soul after death with their healthcare provider. This preliminary study characterizes some of those beliefs. By suggesting questions to ask and responses to give, the study provides healthcare professionals a supportive, knowledgeable way to participate in such discussions.

Numerous examples exist of how disrupting the actions of physiological regulators of blood cell development yields hematologic malignancies. The master regulator of hematopoietic stem/progenitor cells GATA-2 was cloned almost 20 years ago, and elegant genetic analyses demonstrated its essential function to promote hematopoiesis. While certain GATA-2 target genes are implicated in leukemogenesis, only recently have definitive insights emerged linking GATA-2 to human hematologic pathophysiologies. These pathophysiologies include myelodysplastic syndrome, acute myeloid leukemia and an immunodeficiency syndrome with complex phenotypes including leukemia. As GATA-2 has a pivotal role in the etiology of human cancer, it is instructive to consider mechanisms underlying normal GATA factor function/regulation and how dissecting such mechanisms may reveal unique opportunities for thwarting GATA-2-dependent processes in a therapeutic context. This article highlights GATA factor mechanistic principles, with a heavy emphasis on GATA-1 and GATA-2 functions in the hematopoietic system, and new links between GATA-2 dysregulation and human pathophysiologies.

Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.

BACKGROUND: Beliefs about what happens at the time of death surely affect a patient's whole dying experience and could help guide end-of-life care. Yet virtually no research describes those beliefs. This exploratory study begins the descriptive process. METHODS: Assuming culture is key, we interviewed 26 Mexican-American (MA), 18 Euro-American (EA), and 14 African-American (AA) inpatients about their beliefs concerning what happens at the time of death. RESULTS: One belief, that death separates the dead from the living, was widespread. Majorities of all 3 ethnic group samples and of 5 of the 6 gender subsamples expressed this belief, saying the dead “go” or “leave” from this life. Other beliefs differed by ethnic group or gender. For example, more EAs (50%) than others said death is a momentary event, and more MAs (35%) than others said death involves “being taken” by an external force (always God or Jesus). Furthermore, considerably more EA women (45%) than others said some senses persist after death. In contrast, the physiologic signs that participants cited as defining the exact time of death varied from individual to individual with no ethnic or gender pattern, and no one sign predominated. CONCLUSIONS: A few beliefs about what happens at the time of death may characterize Americans in general; many other beliefs may characterize only certain ethnic groups, genders, or individuals. To identify such beliefs and to use them to guide end-of-life care, hospitalists and other health professionals may have to elicit them directly from patients or survivors. Journal of Hospital Medicine 2012. © 2011 Society of Hospital Medicine

Assessment of F-MuLV-induced tumorigenesis reveals new candidate tumor genes including Pecam1 , St7 , and Prim2

The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation.

Leptin is produced predominantly by white adipocytes and in adults it regulates both appetite and energy expenditure but its role in the neonate remains to be fully established. The aim of this, the first study of leptin administration to Meishan piglets, was to examine the effects of chronic leptin administration to neonatal pigs on their endocrine profile, growth and development. Six Meishan sows gave birth normally at term and 6 pairs of siblings (n = 12), matched by birth weight and gender (male, n = 6; female, n = 6) were randomly allocated to leptin (L: n = 6) or placebo (P: n = 6) administration groups. Piglets remained with their mother throughout the study and from day 3 to 8 of neonatal life each pig received either 4 µg ml–1 kg–1 body weight recombinant human leptin or a saline placebo. Plasma concentrations of key hormones and metabolites were determined in conjunction with messenger RNA (mRNA) for leptin, which was assessed by PCR. Recombinant leptin treatment improved growth performance and promoted skeletal growth in favour of adipose tissue accretion. Circulating plasma leptin concentrations were higher on days 4 and 7 in L pigs. Leptin administration altered the endocrine profile of the neonatal pig, although these changes were not maintained. There were no relationships between plasma leptin and body weight or mRNA leptin abundance, irrespective of treatment. Chronic leptin administration appeared to have a beneficial influence on growth rate and body conformation, which may in part be attributed to alterations in metabolism and nutrient partitioning.

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

Background Although it has received little study, gender may significantly affect patients' attitudes about advance care planning. Methods We asked 26 Mexican American (14 male, 12 female), 18 European American (7 male, 11 female), and 14 African American (7 male, 7 female) inpatients for their attitudes about advance care planning and dying. Coders of different ethnicities and genders performed independent, blinded content analyses of responses. Results The interviews identified 40 themes. Five, including "Advance directives (ADs) improve the chances a patient's wishes will be followed," characterized both genders of all 3 ethnic groups. Although no individual themes distinguished the genders across ethnic groups, 3 meta-themes—or clusters of related themes—did. Men's end-of-life wishes addressed functional outcome alone, but women's wishes addressed other factors, too. Men felt disempowered by the health system, but women felt empowered. Men feared harm from the system, but women anticipated benefit. Each ethnic group expressed these gender differences uniquely. For example, most Mexican American men preferred death to disability, believed "the health care system controls treatment," and wanted no "futile" life support. In contrast, most Mexican American women expressed wishes only about care other than life support (especially about when and where they wanted to die), believed ADs "help staff know… (such) wishes," and trusted the system to "honor (written) ADs." Conclusion Core cultural attitudes observed in both genders of 3 ethnic groups may extend to all Americans. Although core attitudes may support advance care planning for many Americans, health professionals should consider tailoring it to other, ethnic- and gender-specific attitudes.

Objective Several personality traits have been associated with anorexia nervosa (AN) in treatment-seeking samples of patients. The current study used a population-based sample to compare the personality characteristics of women with AN who sought treatment versus those who did not. Method Participants included 27 (14 treatment-seekers, 13 non-treatment-seekers) women with threshold or subthreshold AN and 273 (64 treatment seekers, 209 non-treatment seekers) comparison women from the Minnesota Twin Family Study. Personality was assessed with the higher-order factors and primary scales of the Multidimensional Personality Questionnaire (MPQ). Results Non–treatment-seeking women showed lower levels of negative emotionality, stress reaction, and alienation than treatment-seeking women. Discussion These results suggest that personality deviations may be overestimated in treatment-seeking samples. © 2004 by Wiley Periodicals, Inc.

Overexpression of ErbB2 and ErbB4 receptors in breast cancers may be accompanied by contrasting clinical outcomes. To investigate the molecular mechanisms contributing to these differences, we undertook a comparative study of gene expression regulated by the two receptors. Agonistic antibodies were employed to activate ErbB2 and ErbB4 in isolation from the other ErbBs in breast cancer cells. Gene expression profiling using a 16 755-gene oligonucleotide array was performed to identify transcriptional targets of receptor activation. Our results indicate that, in the same cell line, ErbB2 and ErbB4 activation influence gene transcription differentially. Although there are genes that are regulated by signaling from both receptors, there are also receptor-specific targets that are preferentially regulated by each receptor. We further show that two ligands acting via the same receptor homodimer may activate different subsets of genes. Many of the induced genes are hitherto unidentified targets of ErbB signaling. These include ErbB4 targets EPS15R, GATA4, and RAB2 and ErbB2-activated HRY/HES1 and PPAP2A. Targets of ErbB2 homodimer signaling may be especially important as markers in breast cancer, where ErbB2 homodimerization mediated by overexpression and ligand-independent activation is common.

The nonsteroidal anti-inflammatory drug aspirin and the spice curcumin retard adenoma formation when administered long-term to Apc(Min/+) mice, a model of human familial adenomatous polyposis coli. Both agents interfere with cyclooxygenase activity. When aspirin is administered to Apc(Min/+) mice only postweaning, but not before, it is inefficacious, while curcumin given postweaning is active. Here the hypothesis was tested that dietary aspirin (0.05%) or curcumin (0.2%) prevent or delay adenoma formation in offsprings when administered to Apc(Min/+) mothers and up to the end of weaning, but not afterwards. Whereas curcumin was without effect when administered in this way, aspirin reduced numbers of intestinal adenomas by 21%. When aspirin given up to the end of weaning was combined with curcumin administered from the end of weaning for the rest of the animals' lifetime, intestinal adenoma numbers were reduced by 38%. The combination was not superior to intervention postweaning with curcumin alone. These results show that aspirin exerts chemopreventive activity in the Apc(Min/+) mouse during tumour initiation/early promotion, while curcumin is efficacious when given at a later stage of carcinogenic progression. Thus, the results suggest that in this mouse model aspirin and curcumin act during different 'windows' of neoplastic development.

We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.

Breast cancer struck K., a distant cousin of mine, at age 42. She underwent initial mastectomy with nodal irradiation and then aggressive chemotherapy after the cancer recurred 11 years later. When chemotherapy could not control her cancer, K.’s oncologist recommended hospice care. The little information K. shared with me at that time indicated the oncologist had not committed many common mistakes in disclosing terminal cancer. He had not avoided the disclosure altogether, 1 sugarcoated it with ambiguities or euphemisms, or encouraged unrealistic hopes for miraculous cures. Instead, he had discussed candidly the chemotherapy’s failure, the cancer’s resulting incurability, and K.’s likely short survival. 2

A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias.

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.

The inter-rater and test-retest reliabilities, and internal consistencies of the Reiss Screen for Maladaptive Behavior (Reiss 1987) were evaluated on a random sample of adults with moderate through profound mental retardation living in an institutional setting. Generally, the Reiss Screen showed good inter-rater reliability, modest to good test-retest and good internal consistency. This suggests that the Reiss Screen has moderate to good psychometric robustness.

The ecotropic virus integration site 1 (EVI1) transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis. Specifically, we identified EVI1 directly binds to and downregulates the master myeloid differentiation gene Cebpe and several of its downstream gene targets critical for terminal myeloid differentiation. We also found EVI1 binds to and downregulates Serpinb2 as well as numerous genes involved in the Jak-Stat signaling pathway. Finally, we identified decreased expression of several ATP-dependent P2X purinoreceptors genes involved in apoptosis mechanisms. These findings provide a foundation for future study of potential therapeutic gene targets for EVI1-induced leukemia.

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole–imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure–function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1–7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole–imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.

This article reports the investigation and control measures undertaken following the identification of a toxigenic strain of Cornyebacterium diphtheriae var gravis, designated ribotype Minsk , in a partially vaccinated teenager born in the United Kingdom with no recent history of travel or known contact with a case of diphtheria or a carrier. This case highlights the need for ongoing work to improve vaccine uptake rates to ensure children receive all scheduled vaccinations.

Ruthenium complexes [1-3] with oxalato, amido and pyridine ligands have been synthesized and characterized. Biological tests showed that the ruthenium complexes 2 and 3 have modest cytotoxicity on both murine cell line NIH3T3 and human cancer colon cell lines HCT116 and HT29 while complex 1 has no obvious growth defect. We further tested why complexes 2 and 3 exhibit modest cytotoxicity. Gel electrophoresis analysis demonstrates that complex 3 has the ability to cleave double-stranded DNAs in a dose-dependent manner. In contrast, complex 2 does not have detectable DNA cleavage activity. Additionally, real-time PCR analysis suggests that the observed cell growth inhibition or death could be due to the altered gene expression in the processes of cell cycle and apoptosis/necrosis caused by the addition of complexes 2 and 3. Furthermore, cell death by DNA fragmentation assay indicates that the addition of these two complexes may cause cell necrosis rather than apoptosis.

A new automated immunoassay low-mid volume (or = 250 immunoassays/day) chemiluminescent analyzer, Abbott Architect i1000sR, was evaluated by seven laboratories around the world (4 in Europe, one each in Canada, Japan, and the U.S.A.) to demonstrate equivalent performance for key operating characteristics (e.g., precision, turn around time, limit of detection, functional sensitivity, and linearity).The laboratories followed standard protocols to assess precision, limit of detection (LoD), functional sensitivity, assay linearity, method comparison, and sample carryover. Turn around time for three stat assays (beta-hCG, BNP, and CK-MB) and the time required to complete workloads of 50 and 100 tests with a mixture of 75% routine tests and 25% stat tests was also evaluated.Total precision was typically5% CV for nine immunoassays. Analytical performance met design goals and demonstrated equivalency to package insert data for assays on market and in use for an existing high volume immunoassay system. Stat turn around times were consistent with the fixed analytical time of 15.6 minutes and met the expectations of the laboratories. Measured test throughput ranged from 47 - 54 tests per hour and demonstrated that the analyzer was fit for the intended purpose of supporting a laboratory that performsor = 250 immunoassays per day.A multisite, international analyzer familiarization study is a practical means of confirming that a new instrument meets both a manufacturer's design specifications and users' real world expectations and provides a pragmatic test for the system. The experience of investigators at seven sites around the world indicates that a new fully automated chemiluminescent system is suitable for use.

Wanzer, et al., have noted that “all too frequently, physicians are reluctant to withdraw aggressive treatment from hopelessly ill patients, despite clear legal precedent.” Our collective clinical experience confirms that this reluctance persists among some physicians. In this study, we investigated physicians’ attitudes, knowledge, and reported practices regarding the effects of perceived legal constraints on the abatement of life-sustaining treatment from patients who are clearly dying. A factor in assessing these issues is the concept of defensive medicine-that is, the perception that doctors are being forced to order every possible laboratory test and second opinion, or to continue providing non-beneficial life-sustaining treatment, solely to protect themselves from future legal claims. This perception appears widespread among practicing physicians. However, we believe that defensive medicine represents only part of a complex constellation of factors that comprise physicians’ reluctance to abate treatment. This phenomenon encompasses medical, ethical, legal, social, psychological, and spiritual factors interacting in ways that are not fully understood.

Objective The following comprehensive review describes the evolution of stimulant drug formulations used in the treatment of attention-deficit/hyperactivity disorder (ADHD). Emphasis is placed on the basic and clinical pharmacology of the dl-methylphenidate (MPH) transdermal system (MTS). Methods The pharmacokinetic and pharmacodynamic literature pertaining to MPH and amphetamine enantiomers was reviewed in the context of ADHD therapy and MTS as a treatment option. Results MTS incorporates MPH into an adhesive monolithic matrix, using the free base form of the drug to facilitate transdermal absorption. MTS technology minimizes contact dermatitis by eliminating to need for percutaneous penetration enhancers. After a lag time of approximately 2 h, plasma concentrations of the therapeutic d-MPH isomer become detectable, then continuously rise over the course of the recommended 9 h wear time. Concentrations of l-MPH typically attain 40–50% that of d-MPH (vs. 1–2% following oral MPH). Unauthorized MTS removal poses some misuse liability and over 50% of MTS drug content remains in the discarded system. Conclusions While liquid or chewable MPH formulations overcome potential swallowing difficulties, as do sprinkled once-daily extended-release (ER) MPH products, only MTS addresses swallowing difficulties while also offering a flexible individualized MPH exposure time in a once-daily MPH regimen. Copyright © 2008 John Wiley & Sons, Ltd.

This review will begin with a detail of the revision of the WHO classification, and pathological definitions of Burkitt lymphoma. Over the past several years, molecular understanding of Burkitt lymphoma has improved significantly. Using gene expression profiling, a genomic “signature” of Burkitt lymphoma may be identified, that has fidelity beyond c-myc expression, and the presence of the classical t(8;14). Then, evaluation and therapy of the adult patient with Burkitt lymphoma will be reviewed. Relatively few data exist on optimal therapy of the adult patient with Burkitt lymphoma. Principles of therapy should include high doses of alkylating agents, frequent administration of chemotherapy, and attention to central nervous system (CNS) prophylaxis with high doses of systemic chemotherapy, intrathecal therapy, or both. The outcome of adult patients with Burkitt lymphoma, particularly those over 40 years of age, is inferior to the outcome of younger patients, but may be improving over the past few years. Results from an international collaborative effort, which are helpful in evaluating results of Burkitt lymphoma therapy in adults, will be presented. HIV-associated Burkitt lymphoma, and elderly patients with Burkitt lymphoma, comprise special clinical situations that will be also covered in this review.

Generalist physicians must often counsel patients or their families about the right time to die, but feel ill-prepared to do so. Patient beliefs may help guide the discussions.Because little prior research addresses such beliefs, we investigated them in this exploratory, hypothesis-generating study.Anticipating culture as a key influence, we interviewed 26 Mexican Americans (MAs), 18 Euro-Americans (EAs), and 14 African Americans (AAs) and content-analyzed their responses.Nearly all subjects regardless of ethnic group or gender said God determines (at least partially) a patient's right time to die, and serious disease signals it. Yet subjects differed by ethnic group over other signals for that time. Patient suffering and dependence on "artificial" life support signaled it for the MAs; patient acceptance of death signaled it for the EAs; and patient suffering and family presence at or before the death signaled it for the AAs. Subjects also differed by gender over other beliefs. In all ethnic groups more men than women said the time of death is unpredictable; but more women than men said the time of death is preset, and family suffering signals it. Furthermore, most MA women--but few others--explicitly declared that family have an important say in determining a patient's right time to die. No confounding occurred by religion.Americans may share some beliefs about the right time to die but differ by ethnic group or gender over other beliefs about that time. Quality end-of-life care requires accommodating such differences whenever reasonable.

The shared values, beliefs, and behaviors by which people interpret life events are what we call culture. More than medical science, culture determines how people react to illness and death. Science may determine which drug or surgery best treats the disease, but culture often determines how the health professional best treats a patient with the disease. Culture influences when the patient believes he is ill, which treatments he accepts, and which results he prefers. Because culture surely affects illness outcomes, health professionals must not ignore it.This project received financial support from the Mexican American Medical Treatment Effectiveness Research Center and the Aging Research and Education Center at The University of Texas Health Science Center and from the Ecumenical Center for Religion and Health, San Antonio. Presented in an earlier version at the Seventh Annual David C. Thomasma, Ph.D., Memorial International Bioethics Retreat, Medical Faculty, University of Padova, and Fundazione Lanza, Padova, Italy, 26 June 2004; presented at the Second International Conference on Clinical Ethics Consultation, Basel, Switzerland, 19 March 2005; and accepted for presentation at the national meeting of the Society of General Internal Medicine, New Orleans, Louisiana, 13 May 2005. Erika Blacksher, John Stone, Susan Bagby, and David J. Doukas thoughtfully critiqued earlier drafts of this article. Andrew K. Diehl and Helen P. Hazuda critiqued a research abstract of the material. I thank all these people for their many helpful suggestions.

We conducted a prospective, planned study of argon laser panretinal photocoagulation (PRP) in ischemic central retinal vein occlusion (CRVO) over a 10-year period in 123 eyes. On comparing the lasered eyes versus the nonlasered eyes, there was no statistically significant difference between the two groups in the incidence of development of angle neovascularization (NV), neovascular glaucoma (NVG), retinal and/or optic disc NV, or vitreous hemorrhage, or in visual acuity. Our study, however, did show a statistically significant (P = 0.04) difference in the incidence of iris NV between the two groups, with iris NV less prevalent in the laser group than in the nonlaser group, but only when the PRP was performed within 90 days after the onset of CRVO. The other parameter which showed a statistically significant difference between the two groups was the peripheral visual fields-the laser group suffered a significantly (P less than or equal to 0.03) greater loss than the non-laser group. We discuss the implications of these findings in light of the natural history of ischemic CRVO and of ocular NV. Since the original rationale for advocating PRP in ischemic CRVO was the proven beneficial effect of PRP on ocular NV in proliferative diabetic retinopathy, we also discuss the disparities in the disease process between ischemic CRVO and proliferative diabetic retinopathy and in their responses to PRP.

Using functional magnetic resonance imaging (fMRI), we investigated brain activity elicited by a computer-animated child's actions that appeared consistent and inconsistent with a computer-animated adult's instructions. Participants observed a computer-animated adult verbally instructing a computer-animated child to touch one of two objects. The child performed correctly in half of the trials and incorrectly in the other half. We observed significantly greater activity when the child performed incorrectly compared to correctly in regions of the dorsolateral prefrontal cortex (DLPFC) that have been implicated in maintaining our intentions in working memory and implementing cognitive control. However, no such effects were found in regions of the posterior superior temporal sulcus (posterior STS) that have been posited to interpret other people's behavior. These findings extend the role of the DLPFC in cognitive control to evaluating the social outcomes of other people's behavior and provide important new constraints for theories of how the posterior STS contributes to social cognition.