Role of autocrine mediators in the regulation of embryo viability: lessons from animal models.
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Animals↗
Biomedical subjects
C O'Neill
Publications and source records attributed to C O'Neill.
Endogenous folic acid is essential for normal development of preimplantation embryos.
Preimplantation mammalian embryos develop with a high degree of autonomy. To date, there have been no unequivocal demonstrations of a requirement for vitamins in preimplantation embryo development. Reduced folic acid acts as an important methyl donor in many reactions including the synthesis of thymidine. Thymidine does not accumulate in cells so it might be expected that significant amounts of reduced folate would be required to support the exponential increase in DNA synthesis that occurs during early embryo development. The reduction of folate is catalysed by dihydrofolate reductase (EC 1.5.1.3) which is selectively inhibited by the anti-cancer drug methotrexate. Methotrexate caused a dose-dependent inhibition of cell division in 1-cell, 2-cell and 8-cell mouse embryos with 50% inhibition of division occurring at concentrations of 1-10 microM. At a concentration of 0.1 microM only minimal inhibition of the initial cell division occurred, but continuous culture in this concentration of methotrexate completely inhibited further cell divisions. This suggests that most of the exogenous store of reduced folates was used in the first round of cell division. The effects of methotrexate were apparently primarily due to thymidine starvation, since a 10-fold excess of thymidine over methotrexate in culture media reversed the inhibition of development. Supplementing media with folic acid had no beneficial effect on the rate at which zygotes produced by in-vitro fertilization developed to the blastocyst stage. It is concluded that the development of the early embryo has an absolute requirement for reduced folate for thymidine synthesis which is met entirely by endogenous sources.
Animals↗
Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro.
Autocrine mediators released by the early embryo are implicated in the support of embryo development. Their mechanisms and timing of action, however, are uncertain. This study shows that their action is necessary during the 2-cell stage of development, at which time they do not act as classical growth factors, having no impact on the rate of cell-cycle progression. Rather, they act as survival factors, protecting embryos from subsequent cell death. Culture of zygotes for the first 48 h after insemination (but not the first 24 h only) at a high concentration of 1 embryo/microl before transfer to a concentration of 1 embryo/100 microl significantly improved development compared with culture at the lower concentration for 120 h. By contrast, if the initial 48 h of culture was at 1 embryo/100 microl, subsequent culture at 1 embryo/microl caused no improvement. Platelet-activating factor (PAF) supplementation (0.186 microM) of media for the 48-h period after insemination (but not 24 h only) improved the development of embryos cultured at low embryo concentrations (1 embryo/10 microl) even when transferred to PAF-free media for the subsequent 72-h culture period. The ability of PAF to mimic the effects of higher embryo density implicates embryo-derived PAF as one of the autocrine mediators stimulating early embryo development. The cell death that occurred at low embryo concentration had a mixed phenotype: some cells had lost membrane integrity whereas others had intact membranes but punctate or fragmented chromatin. It is concluded that the action of autocrine diffusible factors, including PAF, are necessary during the 2-cell stage for subsequent survival and normal development of embryos to the blastocyst stage.
Animals↗
Costs and effects associated with a community pharmacy-based smoking-cessation programme.
OBJECTIVE: The aim of the study was to determine the costs and effects associated with a community pharmacy-based smoking-cessation programme in Northern Ireland, using the perspective of the payer in the main analysis. DESIGN AND SETTING: Data from a pilot study conducted in 2 community pharmacies in Northern Ireland were used as the basis of the current study, which examined the cost effectiveness of a formal counselling programme for smoking cessation by community pharmacists throughout Northern Ireland. A number of assumptions were made in the baseline analysis (e.g. annual rate of smoking cessation in the absence of the programme; lifetime relapse rate), and these were varied in the sensitivity analysis. PATIENTS AND PARTICIPANTS: The pilot study upon which the main analysis was based was carried out in 2 Belfast pharmacies over a 2-year period. 52 people entered the smoking-cessation programme (group 1), 48 bought nicotine gum and gave their address so that additional information could be sent and they could be followed-up (group 2), and 60 people who expressed a wish to stop smoking were chosen on the basis that they matched, by age, gender, social status and disease status, those in group 1. Thirty-five of those in group 1 requested to use nicotine gum. A statistically significant difference (p < 0.01) was found in cessation rates between intervention and control patients. INTERVENTIONS: The Pharmacists Action on Smoking (PAS) model was the only active intervention used in the study. The model was developed by the PAS group in association with the National Pharmaceutical Association (NPA) in the UK in 1994, and was designed specifically for use by community pharmacists to provide advice and motivation to help smokers stop smoking. The 4-stage model involves a written 'contract' between the patient and pharmacist (including a 'stop date'), and a series of brief counselling meetings over a period of approximately 6 months. MAIN OUTCOME MEASURES AND RESULTS: Our findings indicate that the cost per life-year saved when using the PAS programme ranges from 196.76 pounds sterling (Pounds) to 351.45 Pounds for men and from 181.35 Pounds to 772.12 Pounds for women (1997 values), depending on age. This compares favourably with other disease prevention medical interventions such as screening for hypertension or hypercholesterolaemia. CONCLUSIONS: These findings provide an argument for adoption and remuneration of the PAS model in the community pharmacy (primary healthcare) setting.
Cost-Benefit Analysis↗
Amyloid precursor protein heat shock response in lymphoblastoid cell lines bearing presenilin-1 mutations.
The amyloid precursor protein (APP) gene promoter contains a heat shock element. An abnormal APP heat shock response could increase accumulation of A beta, the APP metabolite found in Alzheimer's disease amyloid plaques. Since A beta production is affected by presenilin-1 (PS-1) mutations, we investigated whether basal APP levels or response to heat shock were altered in lymphoblastoid cell lines from 8 PS-1 mutation-bearers and 9 control members of Alzheimer's disease families. Lymphoblastoid cell lines were incubated at 42 degrees C for 35 min and allowed to recover at 37 degrees C for 1, 3, 8, 24 and 48 h. APP mRNA levels, quantified using RNA-RNA solution hybridisation, increased significantly at 1 and 3 h post-heat shock to between 123% and 163% of pre-heat shock (0 h) levels and returned to normal by 8 h. Semi-quantitative Western immunoblotting of cell lysates using the 22C11 antibody detected two major bands, migrating at approximately 145 and approximately 120 kDa. Band optical densities increased significantly at 3 h to approximately 155% of 0 h levels, following the increase in APP mRNA levels and showing a similar reversibility. APP mRNA and protein responses were comparable in the PS-1 mutation-bearing and control cell lines. This study shows that both APP mRNA and protein are induced in lymphoblastoid cell lines following heat shock and that this response is not affected by PS-1 mutations which are pathogenic for Alzheimer's disease.
Alzheimer Disease↗
G protein subunit levels in fibroblasts from familial Alzheimer's disease patients: lower levels of high molecular weight Gs alpha isoform in patients with decreased beta-adrenergic receptor stimulated cAMP formation.
Abnormalities in G protein linked signal transduction pathways have been detected in fibroblasts from individuals with familial and sporadic Alzheimer's disease. The present study used Gs alpha, Gi alpha, Gq alpha and Go alpha G protein subunit antisera, immunoblotting and densitometry to quantify levels of these proteins in control fibroblasts and in fibroblasts from individuals with familial Alzheimer's disease (FAD). The FAD fibroblasts were from individuals with the APPK670N,M671L mutation, different presenilin 1 (PS1) mutations and one fibroblast cell line from an individual with FAD of unknown genetic aetiology. Results revealed a significant reduction in the large Gs alpha subunit in fibroblasts with the PS1 mutations and in the fibroblast cell line of unknown genetic aetiology, when compared to control levels. This decrease was not apparent in the APPK670N,M671L FAD fibroblasts. Immunoreactivity for Go alpha was not detected in any of the fibroblast cell lines. No differences were observed in Gi alpha or Gq alpha levels when comparing any of the control and Alzheimer's disease fibroblast groups. WE conclude that with the exception of decreased levels of the large Gs alpha subunit, gross alterations in the levels of the Gi alpha, Gq alpha and Go alpha are not associated with the G protein-coupled signal transduction disturbances described previously for some of these FAD fibroblasts.
Alzheimer Disease↗
Studies of the nature of the binding by albumin of platelet-activating factor released from cells.
This report confirms that human umbilical vein endothelial cells activated by A23187 produce platelet-activating factor (PAF) (22.4 +/- 9.9 ng/10(6) cells/h; mean +/- S.E.). A proportion of the PAF produced (56%) was released by cells into the medium. The PAF released, however, was not detected without prior organic extraction, and the method of organic extraction was critical for detection. Extraction with 80% ethanol was not successful, but a modified methanol/chloroform extraction method was. These observations may explain some of the conflicting reports in the literature on release of PAF by activated endothelial cells. The requirements for organic extraction may reflect the nature of cell-released PAF's binding by albumin; it was observed that PAF added to identical media could be detected in a bioassay without the requirement for extraction. Such PAF was also readily degraded by PAF-acetylhydrolase added to media, while PAF released from cells was resistant to such degradation, suggesting that it was released in a "protected" configuration. Stimulation of cells was performed in media with albumin as the only extracellular macromolecule. Limited proteolytic digestion of the albumin with trypsin and pepsin showed that PAF released by cells was located exclusively between amino acids 240 and 386 (domain II), while no synthetic PAF added to media was located on this region. These results are identical to those described for the release of PAF by the early embryo. Albumin exposed to embryos had a higher thiol concentration (0.77 +/- 0.04 micromol of thiol/micromol of albumin; mean +/- S.E.) than control media to which an equivalent amount of synthetic PAF was added (0.59 +/- 0.02 micromol of thiol/micromol of albumin) (measured with Ellman's reagent). Furthermore, albumin from conditioned media was more susceptible to reduction by 10 mM dithiothreitol than control albumin, as assessed by its mobility on PAGE. The protected configuration of released PAF was caused by cell-dependent conformational changes to albumin involving cysteine-cysteine disulfide bonds. Partial reduction with dithiothreitol of albumin exposed to cells resulted in released PAF being able to be detected directly in a bioassay without the requirement for prior organic extraction.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media.
Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF. Thus, effective supplementation of media with PAF requires inactivation of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera. When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10%. The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined. A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid-treatment protocol. The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Issues in cost function specification for neonatal care: the Fordham case.
BACKGROUND: Econometric techniques have been used to examine the relationship between costs of provision, case mix and unit size for health care providers. Estimation of cost functions in a health care context is complicated by poor understanding of the underlying production relationship and the constraints under which production takes place. Different results and policy implications can follow from different model specifications. This underscores the need for care in the construction of such functions and the interpretation of their results. METHODS: Cost and activity data from a study of neonatal care for an English Regional Health Authority are re-examined. Cost functions are estimated using alternative functional forms, and average cost per day is estimated and compared for two of these functions [one produced by Fordham et al. (J Publ Hlth Med 1992; 14(2): 127-130) and the other based more explicitly in economic and econometric theory]. RESULTS: It is shown that estimates of average cost per day are sensitive to model specification. Such are the differences in cost that significantly different policy implications could follow from the different models. CONCLUSION: We conclude that care must be taken in the construction and estimation of cost functions and that the assumptions upon which they rest be made explicit so that results can be properly interpreted.
Cost Allocation↗
Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro.
The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.
Animals↗
Expression of immunoglobulin superfamily cell adhesion molecules on murine embryonic stem cells.
The expression of cell adhesion molecules of the Ig superfamily (Ig-CAM) were examined on embryonic stem (ES) cells during culture in vitro. ES cells maintained an undifferentiated phenotype when cultured in the presence of leukemia inhibitory factor (LIF) or with fibroblast feeder cells; > 90% of cells reacted positively to an antibody (ECMA-7) that marks undifferentiated ES cells. Using flow cytometry, high concentrations of ICAM-1, VCAM-1, and NCAM antigens were detected on undifferentiated ES cells, but their specific receptors, Mac-1, LFA-1, and VLA-4, were not detected. There was also no class I or II major histocompatibility complex (MHC) antigen expression. The ICAM-1 expressed was functional, since anti-ICAM-1 significantly (p < 0.0001) blocked ES cell-lymphocyte binding. Ig-CAM and MHC-1 expression on undifferentiated ES cells was not up-regulated by treatment of cells with interferon-gamma (IFN-gamma), tumor necrosis factor alpha, or flavivirus infection, agents that up-regulate these molecules in other embryonic cell types. Twelve hours after LIF withdrawal, ICAM-1 and NCAM expression decreased significantly, while VCAM-1 was undetectable. However, morphology and ECMA-7 expression remained unchanged. Similar patterns of expression were seen on ES cells maintained on fibroblast feeder cells. This suggests that LIF or other cytokines may maintain the expression of Ig-CAMs on undifferentiated cells. Differentiation was induced by dimethyl sulfoxide treatment for 14 days. Cells changed from a colony-forming to a monolayer morphology, and approximately 60% of the cell population no longer expressed ECMA-7. In these cells, VCAM-1 was undetectable and ICAM-1 and NCAM had declined to low levels. In these differentiated cells, ICAM-1 and MHC-1 were inducible by IFN-gamma. This study suggests that the pattern of expression of the Ig-CAMs in ES cells may have a role in defining the phenotype of differentiated and undifferentiated cells.
Animals↗
Partnership in chronic health care: care management as an integral part of the paediatric hospital system.
Care management for children with complex care needs in the hospital setting is not just about care managers developing and maintaining partnerships between hospital and community professionals, children and their families. It also requires organizational commitment to a system which is patient centred, recognizes parental expertise and offers clinical skills which are co-ordinated across specialty areas.
Australia↗
The role of albumin in the release of platelet-activating factor by mouse preimplantation embryos in vitro.
Platelet-activating factor (PAF) produced by embryos remained associated with mouse four-cell embryos after culture in vitro and was also released into the medium. The release of PAF into medium required albumin as a media supplement and the amount of PAF released increased (P < 0.05) with increasing albumin concentration. There was a trend for the amount of PAF remaining associated with embryos to decrease as the extracellular albumin concentration increased. The association of released PAF with albumin was confirmed by size fractionation with size exclusion membranes and high performance gel filtration, and by affinity chromatography (Cibacron blue and anti-BSA) and native PAGE. PAF released from embryos was not degraded by serum PAF:acetylhydrolase (PAF:AH; E.C. 3.1.1.47) after exposure for 24 h to the serum in vitro, while an equivalent concentration of synthetic PAF added to identical media was readily degraded, suggesting that PAF released from the embryo was protected from PAF:AH action. However, when the medium was subjected to organic extraction by the Bligh-Dyer (methanol/chloroform) method and the resulting extract added to equivalent media, embryo-derived PAF was readily degraded by PAF:AH. Furthermore, PAF in embryo-conditioned medium (30 two-cell embryos for 24 h) could not be detected after direct assay of the culture medium by radioimmunoassay or bioassay (platelet aggregation in vitro), yet after extraction, purification and addition to medium with BSA, the embryo-derived PAF was readily detected in either assay. To determine whether the different behaviour of synthetic PAF and embryo-derived PAF resulted from differences in the nature of their binding to albumin, the location to which PAF bound was assessed by limited proteolytic digestion of albumin. Digestion with pepsin or trypsin showed that embryo-derived PAF was located exclusively between amino acids 240 and 386 (domain II) of albumin. Most synthetic PAF added to equivalent medium not exposed to embryos was not at this location, suggesting that PAF released from embryos bound to a site on albumin not generally accessible to synthetic PAF added to similar media.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
CTP:phosphocholine cytidylyltransferase activity in the preimplantation mouse embryo.
Cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 microgram lysophosphatidylethanolamine ml-1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 +/- 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min-1 (mean +/- SEM). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 +/- 0.05 fmol CDP-choline produced per embryo min-1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by alpha-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Dysarthric speech: a comparison of computerized speech recognition and listener intelligibility.
The purpose of this study was to identify and compare the recognition of dysarthric speech by a computerized voice recognition (VR) system and non-hearing-impaired adult listeners. Intelligibility "functions" were obtained for six dysarthric speakers who varied in severity and six age- and gender-matched controls. Speakers produced 70-item word lists over 5 sessions. VR using the IBM VoiceType and perceptual judgment scores were obtained and functions plotted by session. Data indicate that computerized recognition of both dysarthric and nonimpaired speech was characterized by initially steep increases in correct recognition with more gradual increases noted during the second through fifth sessions. Perceptual recognition by non-hearing-impaired adults indicates generally stable intelligibility scores over time. Severity of dysarthria did appear to influence recognition of target stimuli. Implications of these data to the application of computerized VR technology are presented.
Adolescent↗
Receptor-effector coupling dysfunctions in Alzheimer's disease.
There is now good evidence that in the AD brain, a number of neurotransmitter effector systems are defective. Such abnormalities include defective G, protein and protein kinase C function as well as a drastically reduced level of receptors for the second messenger Ins(1,4,5) P3. Such changes are probably not restricted to the late stages of the disease, and are found in regions of the brain that show little histopathological abnormality, such as the cerebellum. Whether these changes precede or are secondary to primary histopathological changes such as beta-amyloid deposition is not as yet clear. What is clear, however, is that such signal transduction abnormalities are likely to negate therapeutic benefits in clinical strategies based upon the tenet of neurotransmitter replacement.
Alzheimer Disease↗
Neurotransmitter receptor/G-protein mediated signal transduction in Alzheimer's disease brain.
Recent evidence suggests that the neurochemical pathology of Alzheimer's disease includes severe disruptions of the neurotransmitter receptor/G-protein mediated phosphatidylinositol hydrolysis and adenylyl cyclase signal transduction pathways. The present article briefly reviews evidence from postmortem studies describing disruptions to these systems and speculates as to the importance of these changes in terms of contributing to disease pathology and limiting the success of neurotransmitter replacement strategies.
Alzheimer Disease↗
PAF released by preimplantation embryos binds to albumin.
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Animals↗