Reproductive factors, exogenous hormone use and risk of hepatocellular carcinoma among US women: results from the Liver Cancer Pooling Project.

BACKGROUND: Hepatocellular carcinoma (HCC) occurs less commonly among women than men in almost all regions of the world. The disparity in risk is particularly notable prior to menopause suggesting that hormonal exposures during reproductive life may be protective. Exogenous oestrogenic exposures such as oral contraceptives (OCs), however, have been reported to increase risk, suggesting that estrogens may be hepatocarcinogenic. To examine the effects of reproductive factors and exogenous hormones on risk, we conducted a prospective analysis among a large group of US women. METHODS: In the Liver Cancer Pooling Project, a consortium of US-based cohort studies, data from 799,500 women in 11 cohorts were pooled and harmonised. Cox proportional hazards regression models were used to generate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of reproductive factors and exogenous hormones with HCC (n=248). RESULTS: Bilateral oophorectomy was associated with a significantly increased risk of HCC (HR=2.67, 95% CI=1.22-5.85), which did not appear to be related to a shorter duration of exposure to endogenous hormones or to menopausal hormone therapy use. There was no association between OC use and HCC (HR=1.12, 95% CI=0.82-1.55). Nor were there associations with parity, age at first birth, age at natural menopause, or duration of fertility. CONCLUSIONS: The current study suggests that bilateral oophorectomy increases the risk of HCC but the explanation for the association is unclear. There was no association between OC use and HCC risk. Examination of endogenous hormone levels in relation to HCC may help to clarify the findings of the current study.

Experimental identification of diffusive coupling using 2D NMR.

Spin relaxation based nuclear magnetic resonance (NMR) methods have been used extensively to determine pore size distributions in a variety of materials. This approach is based on the assumption that each pore is in the fast diffusion limit but that diffusion between pores can be neglected. However, in complex materials these assumptions may be violated and the relaxation time distribution is not easily interpreted. We present a 2D NMR technique and an associated data analysis that allow us to work directly with the time dependent experimental data without Laplace inversion to identify the signature of diffusive coupling between different pores. Measurements on microporous glass beads and numerical simulations are used to illustrate the technique.

Atrophy and programmed cell death of skeletal muscle.

Striated skeletal is subject to nonlethal cycles of atrophy in response to a variety of physiological and pathological stimuli, including: starvation, disuse, denervation and inflammation. These cells can also undergo cell death in response to appropriate developmental signals or specific pathological insults. Most of the insights gained into the control of vertebrate skeletal muscle atrophy and death have resulted from experimental interventions rather than natural processes. In contrast, the intersegmental muscles (ISMs) of moths are giant cells that initiate sequential and distinct programs of atrophy and death at the end of metamorphosis as a normal component of development. This model has provided fundamental information about the control, biochemistry, molecular biology and anatomy of naturally occurring atrophy and death in vivo. The ISMs have provided a good complement to studies in vertebrates and may provide insights into clinically relevant disorders.

Essential genes that regulate apoptosis.

The expression of several genes has been associated with the induction of apoptosis in a wide variety of vertebrate and invertebrate organisms. However, relatively few gene products have been demonstrated to be required for cell death. This review highlights genes that are required for apoptosis and proposes mechanisms by which the proteins encoded by these genes might function.

Voltage-dependent sodium channels in an invertebrate striated muscle.

Striated skeletal muscles from the planktonic arrowworm Sagitta elegans (phylum Chaetognatha) were voltage-clamped. The muscles displayed classical voltage-dependent sodium channels that (i) showed peak transient currents when the membrane was depolarized 90 millivolts from rest, (ii) opened rapidly with peak currents flowing within 0.4 milliseconds at 4 degrees C, (iii) showed voltage-dependent inactivation with 50 percent inactivation at +25 millivolts from rest, and (iv) were blocked by 500 nanomolar tetrodotoxin.

Peptide and steroid regulation of muscle degeneration in an insect.

Two types of cell death occur in the intersegmental muscles of the giant silkmoth Antheraea polyphemus. The first results from a slow atrophy of the fibers, and the second is a rapid, programmed dissolution of the muscle. Both types appear to be mediated by endocrine factors. The slow atrophy is brought about by the decline in the steroid molting hormone 20-hydroxyecdysone and can be prevented with exogenous steroid. The rapid degeneration is triggered by the peptide eclosion hormone, but the sensitivity of the muscle to the peptide depends on the history of exposure of the muscle to 20-hydroxyecdysone.

Activation of polyubiquitin gene expression during developmentally programmed cell death.

Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examined during the developmentally programmed atrophy and degeneration of the intersegmental muscles (ISMs) in the hawk-moth, Manduca sexta. A clone containing nine repeats of the ubiquitin coding sequence was isolated from an ISM cDNA library and was used as a probe to examine polyubiquitin expression during development. When the ISMs became committed to degenerate, polyubiquitin gene expression increased dramatically. Injection of 20-hydroxyecdysone, which delays degeneration in this system, prevented the increase in polyubiquitin mRNA. The expression of polyubiquitin occurred without apparent activation of the cell's heat shock response. These data suggest that ubiquitin plays a role in programmed cell death.

Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Programmed cell death in the Drosophila central nervous system midline.

BACKGROUND: During the development of the central nervous system, large numbers of cells die by programmed cell death. This process requires the activity of specific gene products and subserves functions that include regulating the sizes of interacting cell populations and removing cells that provide transient functions. Resolution of programmed cell death often involves the elimination of dying cell corpses by phagocytic macrophages. In Drosophila, the reaper gene plays a crucial role in mediating programmed cell death; chromosomal deficiencies which remove reaper result in an absence of programmed cell death. We have used a reaper-deficiency mutant strain Df(3R)H99 (or H99), in conjunction with strains containing cell-type-specific markers, to examine the role of programmed cell death in differentiation of the embryonic central nervous system midline. RESULTS: Midline cell death was identified both by the presence of excess midline cells in H99 mutants and by the engulfment of dying midline cells by macrophages in wild-type embryos. These developmental deaths are lineage-specific: prominent midline glial death was observed, while little if any death was detected among the ventral unpaired median neurons. Examination of H99 mutants indicates that cell death is not required for the formation of macrophage precursors, or for their subsequent migration throughout the embryo; however, in the absence of dying cells, macrophage precursors do not exhibit morphological differentiation or phagocytosis. In both wild-type and H99 mutant embryos, a subset of macrophages migrate along the ventral midline. This midline migration is not observed in single-minded mutants, in which ventral midline cells fail to develop. CONCLUSIONS: Programmed cell death plays a crucial role in the development of the central nervous system midline, and dying midline cells are rapidly eliminated by phagocytic macrophages. It seems that the generation of engulfment signals in cells undergoing programmed cell death is downstream of reaper gene function, and that central nervous system midline and/or ventral epidermal cells provide directional cues for migrating macrophages.

Cold thoughts of death: the role of ICE proteases in neuronal cell death.

While there has been extensive work describing the timing, location and probable signals responsible for regulating programmed cell death (PCD) in the nervous system, relatively little is known about the molecular mechanisms that mediate this process. Several investigators have demonstrated that PCD in general, and neuronal PCD in particular, can be inhibited by drugs that arrest RNA or protein synthesis. These data have been interpreted as suggesting that de novo gene expression is required for cells to commit suicide. The general picture emerging from a number of experimental systems is that a variety of proteins can mediate the coupling of extracellular signals to a resident cell-death program. In this model, some of the components required for death are more or less constitutively present in the cell and await lineage-specific signals for their activation. A recent flood of papers has presented convincing evidence that the resident program for apoptosis in numerous cell types works via a series of essential proteases belonging to the CED-3/ICE family.

Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes.

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study we use the Gal4/UAS targeted gene expression system to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. We also show that in both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally we analyzed the function of a truncated Reaper-C protein which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C revealed cell killing activities distinct from full length Reaper, and indicated that the conserved NH2-terminal domain acts in part to modulate Reaper activity.

The RHG motifs of Drosophila Reaper and Grim are important for their distinct cell death-inducing abilities.

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH(2)-terminal Reaper, Hid, Grim (RHG) motif. We have analyzed the importance of the RHG motifs in Reaper and Grim for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicated that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retained an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions were not efficiently blocked by the cell death inhibitors, Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, we identified a second region of sequence similarity in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities.

Trends in diagnostic testing following a national guideline for evaluation of dyspepsia.

BACKGROUND: The extent to which national guidelines affect actual clinical practice is largely unknown. OBJECTIVE: To gauge the impact of a national guideline about dyspepsia on subsequent clinical practice. DESIGN: We compared trends in the rates of the two principal diagnostic tests used to evaluate dyspepsia: upper gastrointestinal tract series (UGI) and endoscopy. METHODS: We used the Health Care Financing Administration's (Baltimore, Md) 100% National Medicare part B file to determine annual counts for the two procedures in each of 10 years (1984 through 1993). To calculate procedure rates, we divided the number of procedures in each year by the total population enrolled in Medicare part B in that year. RESULTS: In 1984, before publication of the guideline, the UGI rate was more than triple the rate of endoscopy (UGI rate, 59 per 1000 beneficiaries; endoscopy rate, 17 per 1000). Subsequent to publication of the guideline in 1985, UGI use slowly decreased while endoscopy utilization slowly increased. The rate of change for both procedures was steady over the 10-year period. Endoscopy rates finally exceeded UGI in 1993 (endoscopy, 37 per 1000; UGI, 36 per 1000). CONCLUSIONS: Despite a prominent national guideline, UGI utilization remains high and is slowly decreasing. Because utilization data before 1984 are incomplete, we cannot isolate the precise impact of the guideline. However, the slow decline in UGI utilization suggests that, at best, the guideline had limited impact on clinical practice. If this response is representative of other guidelines, alternative approaches to change clinical practice will need to be found.

Women's understanding of the mammography screening debate.

BACKGROUND: The fractious public debate over mammography screening recommendations for women aged 40 to 49 years has received extensive attention in medical journals and in the press. OBJECTIVE: To learn how women interpret the mammography screening debate. METHODS: We mailed a survey to a random sample of American women 18 years and older, oversampling women of screening age (40-70 years). Sixty-six percent of women completed the survey (n = 503). MAIN OUTCOME MEASURES: The main outcome measures were women's reactions to the debate, their suggestion for the starting age for mammography screening, and their understanding of the source of the debate. RESULTS: Almost all women (95%) said that they had paid some attention to the recent discussion about mammography screening. Only 24% said the discussion had improved their understanding of mammography, while 50% reported being upset by the public disagreement among screening experts. Women's beliefs about mammography differed from those articulated by experts in the debate. Eighty-three percent believed that mammography had proven benefit for women aged 40 to 49 years, and 38% believed that benefit was proven for women younger than 40 years. Most women suggested that mammography screening should begin before age 40 years, while only 5% suggested a first mammogram should be performed at 50 years or older. In response to an open-ended question about why mammography has been controversial, 15% cited concerns about the potential harms of radiation and another 12% cited questions about efficacy. Nearly half (49%), however, identified costs as the major source of debate (eg, "Health maintenance organizations [HMOs] don't want to pay for mammography"). CONCLUSIONS: Most women paid attention to the recent debate about routine mammography screening for women aged 40 to 49 years, but many believed the debate was about money rather than the question of benefit. Policy makers issuing recommendations about implementation of large-scale mammography screening services need to consider how to effectively disseminate their message.

Treatment and health outcomes of women and men in a cohort with coronary artery disease.

BACKGROUND: Women with coronary artery disease are treated differently than men. Although mortality has been studied, functional outcomes for women and men have not been prospectively compared. METHODS: The Manitoba Health Reform Impact Study used hospital databases to identify all residents aged 45 years and older in Manitoba who were hospitalized for a myocardial infarction between October 1, 1991, and September 30, 1992. Cohort members were interviewed twice, an average of 16 and 25 months after hospitalization. Baseline and follow-up measures included treatments (eg, physician visits, diagnostic testing, revascularization, and cardiac medications), physical health status (physical component summary [PCS] score derived from the Medical Outcomes Study Short Form 36), reinfarction, and mortality. RESULTS: Of the 820 patients who completed the initial survey, 31 died during the follow-up period, and 734 completed the follow-up survey. Data were complete for the primary outcome (PCS score) and all relevant covariates for the 677 patients who were included in this study Women constituted 34% of this cohort. Although women had more physician visits during follow-up, they were less likely to have undergone treadmill testing or angiography (odds ratio, 0.68; 95% confidence interval, 0.46-0.99). Women were equally likely to report taking beta-adrenergic blocking agents, but were less likely than men to report the use of aspirin (odds ratio, 0.69; 95% confidence interval, 0.48-0.98). After adjusting for baseline differences in PCS scores, age, income, social supports, and the levels of angina and dyspnea, the PCS score for women declined by 1.4 points, while the score for men improved by 0.2 points (P = .03). During the follow-up period, reinfarction and mortality rates were low overall, but were not different in men and women. CONCLUSIONS: In this cohort of patients with known coronary artery disease, we found less aggressive treatment of coronary artery disease and less use of aspirin among women than among men during 1 year of observation. After controlling for baseline differences, women with coronary artery disease experienced a more rapid decline in physical health status than did men during 1 year of follow-up.

The novel calcium antagonist Ro 40-5967 limits myocardial infarct size in the dog.

OBJECTIVE: The aim was to compare the infarct limiting effect of Ro 40-5967 (Ro40), a new calcium antagonist with little negative inotropic activity, with that of verapamil and with ischaemic preconditioning, a potent endogenous cardioprotective mechanism. METHODS: Dogs (n = 53) of either sex were subjected to 60 min of coronary occlusion followed by 3 h of reperfusion. Drug treated dogs received either verapamil (1.0 mg.kg-1) or Ro40 (3.0 mg.kg-1) intravenously for 100 min starting 15 min prior to the occlusion. Control dogs received a saline infusion. Ischaemic preconditioning consisted of four 5 min cycles of ischaemia alternating with four 5 min cycles of reperfusion. After 3 h of reflow, hearts were excised and infarct size was measured using tripheyltetrazolium chloride macrochemistry and expressed as percent of the ischaemic area at risk (AAR). To control for variation in infarct size due to variation in collateral blood flow, infarct size among groups was compared using ANCOVA, in which infarct size and collateral blood flow, measured at 30 min of occlusion, were dependent and independent variables, respectively. RESULTS: Transmural collateral blood flow and AAR were not significantly different between any of the groups. Mean infarct size (adjusted by ANCOVA for slight differences in collateral blood flow among groups) in control dogs (n = 13) was 25.9(SEM 3.2)% of the AAR. Both verapamil (n = 11) and Ro40 (n = 9) limited infarct size [14.2(3.2)% AAR and 16.7(2.9)% AAR, respectively; both p < 0.05]. Preconditioning (n = 17) also significantly limited infarct size [8.1(1.8)%; p < 0.01]. CONCLUSIONS: The new calcium antagonist, Ro 40-5967, was as effective as verapamil in limiting infarct size after 60 min of regional ischaemia followed by 3 h of reperfusion, although neither calcium antagonist was as effective as ischaemic preconditioning.

Effect of catecholamine depletion on myocardial infarct size in dogs: role of catecholamines in ischemic preconditioning.

OBJECTIVES: Cardioprotective adaptation to brief periods of ischemia and reperfusion is termed ischemic preconditioning (PC). Limitation of infarct size by preconditioning is associated with marked slowing of ischemic metabolism. The cause of metabolic slowing has not been determined but may involve either pro- or anti-adrenergic mechanisms. Hypothetically, adrenergic stimulation could signal the adaptive response. Alternatively, metabolic slowing during the sustained ischemic challenge could occur through a reduction in beta-adrenergic stimulation. This study was designed to test the role of cardiac norepinephrine (NE) in PC. METHODS: The effect of PC on myocardial infarct size was studied in control dogs and dogs depleted of catecholamines by pretreatment with reserpine (RES; 0.25 mg/kg i.v.). PC was induced by four cycles of 5 min of ischemia and 5 min of reperfusion. Infarcts were produced by 60 min of ischemia and 3 h of reperfusion. Cardiac NE depletion was verified by radioimmunoassay of tissue samples and by absence of hemodynamic response to a tyramine bolus (1.4 mg/kg) administered at the end of each experiment. Infarct size, expressed as percent of area at risk, was controlled for variation in collateral blood flow using analysis of covariance (ANCOVA). RESULTS: Adjusted mean infarct size was 25.5 +/- 3.2% in untreated controls vs. 19.1 +/- 3.3% in RES-treated controls (P = NS). PC limited infarct size in untreated dogs (7.4 +/- 1.8 vs. 25.5 +/- 3.2%; PC vs. control; P < 0.01) but not in RES-treated dogs (15.7 +/- 3.0% vs. 19.1 +/- 3.3%; RES + PC vs. RES; P = NS). Infarct size was larger in dogs with RES + PC than with PC alone, even though there was a trend toward a slight beneficial effect with RES alone. CONCLUSION: The cardioprotective effect of ischemic preconditioning cannot be explained entirely as an anti-adrenergic effect. On the contrary, adrenergic receptor stimulation may be required for the full expression of ischemic preconditioning in canine myocardium.

Risk communication in clinical practice: putting cancer in context.

CONTEXT: Clinicians are increasingly urged-even mandated-to help patients make informed medical decisions by paying more attention to risk counseling. For many, the role of risk counseling is new and unfamiliar. This effort is made more difficult given the practical constraints created by 15-minute visits and competing demands (e.g., patient's chief complaint and institutional needs). OBJECTIVE: We detail a three-part approach for improving risk communication, acknowledging the role of clinicians, patients, and other communicators (i.e., media or public health agencies). PROPOSED APPROACH: Office-based tools to help clinicians do more. We suggest two ways to help make up-to-date estimates of disease risk and treatment benefit easily available during office visits. We propose the development of a comprehensive population database about disease risk and treatment benefit to be created and maintained by the federal government. Educating patients. We propose "Understanding Numbers in Health" a tutorial that reviews basic concepts of probability and their application to medical studies to help people become better critical readers of health information. Guidance for communicators. Finally, we propose a writer's guide to risk communication: a set of principles to help health communicators present data to the public clearly and objectively. CONCLUSION: In addition to tools to help clinicians better communicate risk information, serious efforts to improve risk communication must go beyond the clinic. Efforts that help the public to better interpret health risk information and guide communicators to better present such information are a place to start.

Localization of immunoreactive ubiquitin in the nervous system of the Manduca sexta moth.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissues undergoing programmed cell death (e.g., larval intersegmental muscles in Manduca sexta moths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathetic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells committed to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the abdominal ganglia of Manduca sexta suggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated.

Imaginal cell-specific accumulation of the multicatalytic proteinase complex (proteasome) during post-embryonic development in the tobacco hornworm, Manduca sexta.

The multicatalytic proteinase complex is a multi-subunit, high molecular weight proteinase present in the nucleus and cytoplasm of eukaryotic cells. This catalytic complex is involved in diverse cellular functions as part of the ubiquitin proteolysis system, including non-lysosomal proteolysis, antigen presentation, cell cycle progression, and cell proliferation, and in the programmed death of intersegmental muscles after adult eclosion in the tobacco hornworm moth, Manduca sexta. We have investigated the distribution of the multicatalytic proteinase complex in the central nervous system of this moth. At all stages of post-embryonic development, most cell types exhibited consistent, low levels of cytoplasmic and nuclear immunoreactivity for the multicatalytic proteinase complex. High levels of cell-specific accumulation of the complex were, however, demonstrated in abdominal neurosecretory cells and in imaginal cells in the larval brain, the larval segmental ganglia, and the developing wing discs. Imaginal cells exhibited intense immunoreactivity for the multicatalytic proteinase complex only until the onset of terminal differentiation. Intersegmental muscles undergoing programmed cell death exhibited intense cytoplasmic immunoreactivity for the multicatalytic proteinase, while persisting flight muscles and dying neurons were characterized by basal levels of staining. These staining patterns suggest that the multicatalytic proteinase of Manduca sexta serves multiple functions and is associated with the period of developmental arrest displayed by imaginal cells prior to metamorphosis.