Search

AIM Diagnosing and monitoring vascular activity in giant cell arteritis (GCA) is difficult due to the paucity of specific serological biomarkers. We assessed the utility of 8 novel biomarkers in an inception cohort of newly suspected GCA patients. METHOD Consecutive patients were enrolled between May 2016 and December 2017. Serum was collected within 72 hours of commencing corticosteroids and at 6 months. It was analyzed for levels of intra-cellular adhesion molecule 1, vascular endothelial growth factor (VEGF), pentraxin 3, von Willebrand factor and procalcitonin (5-plex R&D Systems multiplex assay) and interleukin (IL)6, IL12 and interferon-γ (high-sensitivity 3-plex ProcartaPlex multiplex assay). A GCA specific positron emission tomography / computed tomography (PET/CT) scan was performed at enrolment with uptake in each vascular territory graded and summed to derive a total vascular score (TVS). RESULTS For the 63 patients enrolled, 12 (19%) had a final diagnosis of biopsy-positive GCA and a further 9 had a clinical diagnosis of biopsy-negative GCA. None of the 8 biomarkers was significantly higher in GCA patients compared with those with alternative diagnoses, or demonstrated a positive correlation with the PET/CT TVS. This was in contrast to the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) which were higher in the biopsy-positive GCA cohort (P

Mass gatherings play an important role in society, but since the onset of the Covid-19 pandemic, they have generally been restricted in order to mitigate transmission of SARS-CoV-2. The aim of this study was to summarise the evidence regarding the effectiveness of public health measures at preventing the transmission of SARS-CoV-2 at mass gatherings, and hence inform guidance on the organisation of these events. A rapid review was undertaken in Cochrane, Embase (OVID), Medline (OVID), Google, Web of Science and Europe PMC from 1 January 2020 to 3 June 2021. Of the identified 1,624 citations, 14 articles referring to 11 unique studies were included. This rapid review found evidence from 11 studies (involving approximately 30,482 participants) that implementing a range of measures may reduce the risk of SARS-CoV-2 transmission at mass gatherings; however, it is unlikely that this risk can be eliminated entirely. All studies adopted a layered mitigation approach involving multiple measures, which may be more effective than relying on any single measure. The number and intensity of measures implemented varied across studies, with most implementing resource intense measures. Importantly, all included studies were only of 'fair' to 'poor' quality. In conclusion, there is currently limited evidence on the effectiveness of measures to prevent SARS-CoV-2 transmission at mass gatherings. As mass gatherings recommence, continued adoption of known mitigation measures is required to limit the risk of transmission, as well as ongoing research and surveillance to monitor the potential impact of these events on the wider population and healthcare system.

Background Many people with chronic disease have more than one chronic condition, which is referred to as multimorbidity. The term comorbidity is also used but this is now taken to mean that there is a defined index condition with other linked conditions, for example diabetes and cardiovascular disease. It is also used when there are combinations of defined conditions that commonly co-exist, for example diabetes and depression. While this is not a new phenomenon, there is greater recognition of its impact and the importance of improving outcomes for individuals affected. Research in the area to date has focused mainly on descriptive epidemiology and impact assessment. There has been limited exploration of the effectiveness of interventions to improve outcomes for people with multimorbidity. Objectives To determine the effectiveness of health-service or patient-oriented interventions designed to improve outcomes in people with multimorbidity in primary care and community settings. Multimorbidity was defined as two or more chronic conditions in the same individual. Search methods We searched MEDLINE, EMBASE, CINAHL and seven other databases to 28 September 2015. We also searched grey literature and consulted experts in the field for completed or ongoing studies. Selection criteria Two review authors independently screened and selected studies for inclusion. We considered randomised controlled trials (RCTs), non-randomised clinical trials (NRCTs), controlled before-after studies (CBAs), and interrupted time series analyses (ITS) evaluating interventions to improve outcomes for people with multimorbidity in primary care and community settings. Multimorbidity was defined as two or more chronic conditions in the same individual. This includes studies where participants can have combinations of any condition or have combinations of pre-specified common conditions (comorbidity), for example, hypertension and cardiovascular disease. The comparison was usual care as delivered in that setting. Data collection and analysis Two review authors independently extracted data from the included studies, evaluated study quality, and judged the certainty of the evidence using the GRADE approach. We conducted a meta-analysis of the results where possible and carried out a narrative synthesis for the remainder of the results. We present the results in a 'Summary of findings' table and tabular format to show effect sizes across all outcome types. Main results We identified 18 RCTs examining a range of complex interventions for people with multimorbidity. Nine studies focused on defined comorbid conditions with an emphasis on depression, diabetes and cardiovascular disease. The remaining studies focused on multimorbidity, generally in older people. In 12 studies, the predominant intervention element was a change to the organisation of care delivery, usually through case management or enhanced multidisciplinary team work. In six studies, the interventions were predominantly patient-oriented, for example, educational or self-management support-type interventions delivered directly to participants. Overall our confidence in the results regarding the effectiveness of interventions ranged from low to high certainty. There was little or no difference in clinical outcomes (based on moderate certainty evidence). Mental health outcomes improved (based on high certainty evidence) and there were modest reductions in mean depression scores for the comorbidity studies that targeted participants with depression (standardized mean difference (SMD) −2.23, 95% confidence interval (CI) −2.52 to −1.95). There was probably a small improvement in patient-reported outcomes (moderate certainty evidence) although two studies that specifically targeted functional difficulties in participants had positive effects on functional outcomes with one of these studies also reporting a reduction in mortality at four year follow-up (Int 6%, Con 13%, absolute difference 7%). The intervention may make little or no difference to health service use (low certainty evidence), may slightly improve medication adherence (low certainty evidence), probably slightly improves patient-related health behaviours (moderate certainty evidence), and probably improves provider behaviour in terms of prescribing behaviour and quality of care (moderate certainty evidence). Cost data were limited. Authors' conclusions This review identifies the emerging evidence to support policy for the management of people with multimorbidity and common comorbidities in primary care and community settings. There are remaining uncertainties about the effectiveness of interventions for people with multimorbidity in general due to the relatively small number of RCTs conducted in this area to date, with mixed findings overall. It is possible that the findings may change with the inclusion of large ongoing well-organised trials in future updates. The results suggest an improvement in health outcomes if interventions can be targeted at risk factors such as depression, or specific functional difficulties in people with multimorbidity.

The aim of this rapid review is to summarise the evidence on non-contact thermal screening as a method through which to identify cases and reduce the spread of coronavirus disease (Covid-19). The rapid review was conducted in accordance with Cochrane guidelines, with a systematic search of published peer-reviewed articles and non-peer-reviewed pre-prints undertaken from 1 January 2000 up to 7 October 2020. Eleven studies were included. One observational study and two mathematical modelling studies were conducted in the context of the Covid-19 pandemic; the remaining studies were conducted during the influenza A pandemic (H1N1) 2009 (n = 7) or middle east respiratory syndrome (n = 1) pandemics. One systematic review and three rapid reviews were identified and screened for relevant studies. Evidence on the effectiveness of thermal screening contained within this review was limited to points of entry (i.e., airports); thus the applicability to other community settings is uncertain. Thermal screening, implemented as part of a composite of screening measures (self-report of relevant symptoms, contact/travel history), was ineffective in identifying infectious individuals and limiting the spread of disease. Based on limited, low certainty evidence, non-contact thermal screening is ineffective in limiting the spread of Covid-19.

The collection of nasopharyngeal swabs to test for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an invasive technique with implications for patients and clinicians. Alternative clinical specimens from the upper respiratory tract may offer benefits in terms of collection, comfort and infection risk. The objective of this review was to synthesise the evidence for detection of SARS-CoV-2 ribonucleic acid (RNA) using reverse transcription polymerase chain reaction (RT-PCR) tested saliva or nasal specimens compared with RT-PCR tested nasopharyngeal specimens. Searches were conducted in PubMed, Embase, Europe PMC and NHS evidence from December 2019 to 20 July 2020. Eighteen studies were identified; 12 for saliva, four for nasal and two included both specimen types. For saliva-based studies, the proportion of saliva samples testing positive relative to all positive samples in each study ranged from 82.9% to 100%; detection in nasopharyngeal specimens ranged from 76.7% to 100%; positive agreement between specimens for overall detection ranged from 65.4% to 100%. For nasal-based studies, the proportion of nasal swabs testing positive relative to all positive samples in each study ranged from 81.9% to 100%; detection in nasopharyngeal specimens ranged from 70% to 100%; positive agreement between specimens for overall detection ranged from 62.3% to 100%. The results indicate an inconsistency in the detection of SARS-CoV-2 RNA in the specimen types included, often with neither the index nor the reference of interest detecting all known cases. Depending on the test environment, these clinical specimens may offer a viable alternative to standard. However, at present the evidence is limited, of variable quality, and relatively inconsistent.

Summary In this review, we systematically searched and summarized the evidence on the immune response and reinfection rate following SARS‐CoV‐2 infection. We also retrieved studies on SARS‐CoV and MERS‐CoV to assess the long‐term duration of antibody responses. A protocol based on Cochrane rapid review methodology was adhered to and databases were searched from 1/1/2000 until 26/5/2020. Of 4744 citations retrieved, 102 studies met our inclusion criteria. Seventy‐four studies were retrieved on SARS‐CoV‐2. While the rate and timing of IgM and IgG seroconversion were inconsistent across studies, most seroconverted for IgG within 2 weeks and 100% (N = 62) within 4 weeks. IgG was still detected at the end of follow‐up (49‐65 days) in all patients (N = 24). Neutralizing antibodies were detected in 92%‐100% of patients (up to 53 days). It is not clear if reinfection with SARS‐CoV‐2 is possible, with studies more suggestive of intermittent detection of residual RNA. Twenty‐five studies were retrieved on SARS‐CoV. In general, SARS‐CoV‐specific IgG was maintained for 1‐2 years post‐infection and declined thereafter, although one study detected IgG up to 12 years post‐infection. Neutralizing antibodies were detected up to 17 years in another study. Three studies on MERS‐CoV reported that IgG may be detected up to 2 years. In conclusion, limited early data suggest that most patients seroconvert for SARS‐CoV‐2‐specific IgG within 2 weeks. While the long‐term duration of antibody responses is unknown, evidence from SARS‐CoV studies suggest SARS‐CoV‐specific IgG is sustained for 1‐2 years and declines thereafter.

Antihypertensive medication nonadherence is highly prevalent, leading to uncontrolled blood pressure. Methods that facilitate the targeting and tailoring of adherence interventions in clinical settings are required. Group-Based Trajectory Modeling (GBTM) is a newer method to evaluate adherence using pharmacy dispensing (refill) data that has advantages over traditional refill adherence metrics (e.g. Proportion of Days Covered) by identifying groups of patients who may benefit from adherence interventions, and identifying patterns of adherence behavior over time that may facilitate tailoring of an adherence intervention. We evaluated adherence to antihypertensive medication in 905 patients over a 12-month period in a community pharmacy setting using GBTM, identifying three subgroups of adherence patterns: 52.8%, 40.7%, and 6.5% had very high, high, and low adherence, respectively. However, GBTM failed to demonstrate predictive validity with blood pressure at 12 months. Further research on the validity of adherence measures that facilitate interventions in clinical settings is required.

The complexity and heterogeneity of patients with multimorbidity and polypharmacyrenders traditional disease-oriented guidelines often inadequate and complicates clinical decision making. To address this challenge, guidelines have beendeveloped on multimorbidity or polypharmacy. To systematically analyse theirrecommendations, we conducted a systematic guideline review using the Ariadneprinciples for managing multimorbidity as analytical framework. The informationsynthesis included a multistep consensus process involving 18 multidisciplinaryexperts from seven countries. We included eight guidelines (four each onmultimorbidity and polypharmacy) and extracted about 250 recommendations. Theguideline addressed (i) the identification of the target population (riskfactors); (ii) the assessment of interacting conditions and treatments: medicalhistory, clinical and psychosocial assessment including physiological status and frailty, reviews of medication and encounters with healthcare providershighlighting informational continuity; (iii) the need to incorporate patientpreferences and goal setting: eliciting preferences and expectations, the processof shared decision making in relation to treatment options and the level ofinvolvement of patients and carers; (iv) individualized management: guidingprinciples on optimization of treatment benefits over possible harms, treatmentcommunication and the information content of medication/care plans; (v)monitoring and follow-up: strategies in care planning, self-management andmedication-related aspects, communication with patients including safetyinstructions and adherence, coordination of care regarding referral and dischargemanagement, medication appropriateness and safety concerns. The spectrum ofclinical and self-management issues varied from guiding principles to specificrecommendations and tools providing actionable support. The limited availability of reliable risk prediction models, feasible interventions of proveneffectiveness and decision aids, and limited consensus on appropriate outcomes ofcare highlight major research deficits. An integrated approach to bothmultimorbidity and polypharmacy should be considered in future guidelines.

Objective To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model. Methods Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified. Results No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA. Conclusion Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the effects of family‐based interventions to improve outcomes in people with type 2 diabetes mellitus and to consider, where possible, which components of the interventions are most effective.

Background Gestational alcohol exposure causes lifelong physical and neurocognitive deficits collectively referred to as fetal alcohol spectrum disorders (FASDs). Micronutrient deficiencies are common in pregnancies of alcohol-abusing women. Here we show the most common micronutrient deficiency of pregnancy—iron deficiency without anemia—significantly worsens neurocognitive outcomes following perinatal alcohol exposure. Methods Pregnant rats were fed iron-deficient (ID) or iron-sufficient diets from gestational day 13 to postnatal day (P) 7. Pups received alcohol (0, 3.5, 5.0 g/kg) from P 4 to P 9, targeting the brain growth spurt. At P 32, learning was assessed using delay or trace eyeblink classical conditioning (ECC). Cerebellar interpositus nucleus (IPN) and hippocampal CA1 cellularity was quantified using unbiased stereology. Results Global analysis of variance revealed that ID and alcohol separately and significantly reduced ECC learning with respect to amplitude (ps ≤ 0.001) and conditioned response [CR] percentage (ps ≤ 0.001). Iron and alcohol interacted to reduce CR percentage in the trace ECC task (p = 0.013). Both ID and alcohol significantly reduced IPN (ps

Aims The aim was to describe the utilization of antidiabetic agents, in terms of persistence and regimen change, in the management of a cohort of newly treated type 2 diabetes patients and to investigate associated socio-demographic and treatment factors. Methods A population-based retrospective cohort study was conducted using the national pharmacy claims database in Ireland. Subjects were analyzed for persistence and regimen change. Cox proportional hazards regression examined associations of socio-demographic and treatment factors on treatment patterns. Hazard ratios (HR) and 95% CIs are presented. Results A total of 20947 subjects were identified in the study over a 2 year period. Most were initiated on metformin (76%) or sulphonylureas (22%) and 77% were persistent with therapy 12 months after initiation. The likelihood of non-persistence was significantly lower in the youngest (40–49 years) age groups (reference 60–69 years) (HR 1.62, 95% CI 1.42, 1.84) and those on sulphonylureas (HR 1.49, 95% CI 1.36, 1.64). The likelihood of receiving a regimen change was significantly lower in the older (80+ years) age groups (HR 0.63, 95% CI 0.56, 0.71), females (HR 0.91, 95% CI 0.86, 0.95), and those with pre-existing CVD (1 vs. 0 CVD medicines) (HR 0.82, 95% CI 0.74, 0.90), and higher in those on sulphonylureas (HR 1.83, 95% CI 1.73, 1.94). Conclusions Type of treatment, pre-existing CVD and demographic factors are shown to be associated with the observed treatment patterns. Guideline recommended agents were widely used on treatment initiation though a substantial minority were not initiated on the recommended first line agent. Use of guideline recommended agents was not as evident during treatment progression. Further optimization of initial and subsequent antidiabetic agent prescribing may be possible.

Objective To explore the involvement of protease-activated receptor 1 (PAR-1) and PAR-2 in the pathologic processes of osteoarthritis (OA) and to identify the cells/tissues primarily affected by ablation of PAR-1 or PAR-2 in mice. Methods OA was induced in the joints of wild-type (WT), PAR-1(+/+) , PAR-1(-/-) , and PAR-2(-/-) mice by destabilization of the medial meniscus (DMM), and scores of histologic features (cartilage aggrecan loss and erosion, subchondral bone sclerosis, osteophytes, and synovitis) were compared at 1, 4, and 8 weeks post-DMM. The effects of PAR ablation on cartilage degradation and chondrocyte metalloproteinase expression/activity were studied in cultures of mouse femoral head tissue with or without interleukin-1α (IL-1α). At 1 week post-DMM, synovial expression of cytokines and metalloproteinase genes was measured by reverse transcription-polymerase chain reaction, and populations of inflammatory cells were quantified by flow cytometry. Results Deletion of PAR-2, but not that of PAR-1, in mice significantly delayed the progression of cartilage damage and inhibited subchondral bone sclerosis following DMM. There was no inhibitory effect of PAR-1 or PAR-2 ablation on IL-1α-induced cartilage degradation or chondrocyte metalloproteinase expression/activation. A low but significant level of synovitis persisted in mice subjected to DMM compared to that in control mice subjected to sham surgery, but no differences between the genotypes were seen 4 or 8 weeks post-DMM. One week after DMM, increased synovial expression of proinflammatory cytokines and metalloproteinase genes, along with increased levels of CD4+ T cells, inflammatory monocytes, and activated macrophages, were seen in all genotypes. However, there was a significant reduction in the percentage of activated macrophages in PAR-2(-/-) mice compared to PAR-1(-/-) and WT mice. Conclusion Deletion of PAR-2, but not that of PAR-1, results in a significant decrease in DMM-induced cartilage damage. The chondroprotection in PAR-2(-/-) mice appears to occur indirectly through modulation of extracartilaginous events such as subchondral bone remodeling and synovial macrophage activation, rather than through alteration of chondrocyte catabolic responses.

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Some affected individuals possess distinctive craniofacial deficits, but many more lack overt facial changes. An understanding of the mechanisms underlying these deficits would inform their diagnostic utility. Our understanding of these mechanisms is challenged because ethanol lacks a single receptor when redirecting cellular activity. This review summarizes our current understanding of how ethanol alters neural crest development. Ample evidence shows that ethanol causes the “classic” fetal alcohol syndrome (FAS) face (short palpebral fissures, elongated upper lip, deficient philtrum) because it suppresses prechordal plate outgrowth, thereby reducing neuroectoderm and neural crest induction and causing holoprosencephaly. Prenatal alcohol exposure (PAE) at premigratory stages elicits a different facial appearance, indicating FASD may represent a spectrum of facial outcomes. PAE at this premigratory period initiates a calcium transient that activates CaMKII and destabilizes transcriptionally active β-catenin, thereby initiating apoptosis within neural crest populations. Contributing to neural crest vulnerability are their low antioxidant responses. Ethanol-treated neural crest produce reactive oxygen species, and free radical scavengers attenuate their production and prevent apoptosis. Ethanol also significantly impairs neural crest migration, causing cytoskeletal rearrangements that destabilize focal adhesion formation; their directional migratory capacity is also lost. Genetic factors further modify vulnerability to ethanol-induced craniofacial dysmorphology, and include genes important for neural crest development including shh signaling, PDFGA, vangl2, and ribosomal biogenesis. Because facial and brain development are mechanistically and functionally linked, research into ethanol’s effects on neural crest also informs our understanding of ethanol’s CNS pathologies.

OBJECTIVES: To perform a systematic review of the Probability of Repeated Admission (Pra) score in community-dwelling adults to assess its performance in a range of validation studies in the community setting. DESIGN: Systematic review and meta-analysis. SETTING: Primary and community care. PARTICIPANTS: Community-dwelling older people. MEASUREMENTS: The primary outcome was hospital admission; secondary outcomes were mortality, hospital days, functional decline, other health service use, and costs. RESULTS: Nine validation studies describing 11 cohorts of individuals aged 65 and older were identified. A metaanalysis of the Pra score in five cohorts (8,843 individuals) with comparable and available data revealed good discrimination performance (summary area under the receiver operating characteristic curve 69.7% (standard error 2.8%)). Pooled specificity was high (96%, 95% confidence interval (CI)=95.8–96.7%), indicating that a Pra score of 0.5 or greater effectively rules in the likelihood of admission, but pooled sensitivity was low (12%, 95% CI=10.5–13.6%). Calibration performance was good, with an overall risk ratio of 1.12 (95% CI=0.89–1.42), indicating that the Pra score reliably predicted hospital admissions. CONCLUSION: The Pra score performs well in predicting hospital admission in community-dwelling adults categorized as high risk according to the score. This tool has clinical and healthcare policy utility in terms of targeting elderly people at highest risk of hospital admission, but the low pooled sensitivity of the score indicates that it is not a reliable way of excluding hospital admission in those stratified as low risk.

Background Atrial fibrillation (AF), the most common arrhythmia in clinical practice, is a leading cause of morbidity and mortality. Screening for AF in asymptomatic patients has been proposed as a way of reducing the burden of the disease by detecting people who would benefit from prophylactic anticoagulation therapy before the onset of symptoms. However, for screening to be an effective intervention, it must improve the detection of AF and provide benefit for those detected earlier as a result of screening. Objectives This review aims to answer the following questions. Does systematic screening increase the detection of AF compared with routine practice? Which combination of screening population, strategy and test is most effective for detecting AF compared with routine practice? What safety issues and adverse events may be associated with individual screening programmes? How acceptable is the intervention to the target population? What costs are associated with systematic screening for AF? Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (Ovid) and EMBASE (Ovid) up to 11 November 2015. We searched other relevant research databases, trials registries and websites up to December 2015. We also searched reference lists of identified studies for potentially relevant studies, and we contacted corresponding authors for information about additional published or unpublished studies that may be relevant. We applied no language restrictions. Selection criteria Randomised controlled trials comparing screening for AF with routine practice in people 40 years of age and older were eligible. Two review authors (PM and CT) independently selected trials for inclusion. Data collection and analysis Two review authors (PM and CT) independently assessed risk of bias and extracted data. We used odds ratios (ORs) and 95% confidence intervals (CIs) to present results for the primary outcome, which is a dichotomous variable. As we identified only one study for inclusion, we performed no meta-analysis. We used the GRADE (Grades of Recommendation, Assessment, Development and Evaluation Working Group) method to assess the quality of the evidence and GRADEPro to create a 'Summary of findings' table. Main results One cluster-randomised controlled trial met the inclusion criteria for this review. This study compared systematic screening (by invitation to have an electrocardiogram (ECG)) and opportunistic screening (pulse palpation during a general practitioner (GP) consultation for any reason, followed by an ECG if pulse was irregular) versus routine practice (normal case finding on the basis of clinical presentation) in people 65 years of age or older. Results show that both systematic screening and opportunistic screening of people over 65 years of age are more effective than routine practice (OR 1.57, 95% CI 1.08 to 2.26; and OR 1.58, 95% CI 1.10 to 2.29, respectively; both moderate-quality evidence). We found no difference in the effectiveness of systematic screening and opportunistic screening (OR 0.99, 95% CI 0.72 to 1.37; low-quality evidence). A subgroup analysis found that systematic screening and opportunistic screening were more effective in men (OR 2.68, 95% CI 1.51 to 4.76; and OR 2.33, 95% CI 1.29 to 4.19, respectively) than in women (OR 0.98, 95% CI 0.59 to 1.62; and OR 1.2, 95% CI 0.74 to 1.93, respectively). No adverse events associated with screening were reported. The incremental cost per additional case detected by opportunistic screening was GBP 337, compared with GBP 1514 for systematic screening. All cost estimates were based on data from the single included trial, which was conducted in the UK between 2001 and 2003. Authors' conclusions Evidence suggests that systematic screening and opportunistic screening for AF increase the rate of detection of new cases compared with routine practice. Although these approaches have comparable effects on the overall AF diagnosis rate, the cost of systematic screening is significantly greater than the cost of opportunistic screening from the perspective of the health service provider. Few studies have investigated effects of screening in other health systems and in younger age groups; therefore, caution needs to be exercised in relation to transferability of these results beyond the setting and population in which the included study was conducted. Additional research is needed to examine the effectiveness of alternative screening strategies and to investigate the effects of the intervention on risk of stroke for screened versus non-screened populations.

Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure.WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors, or neurons. Cells along this progression were cultured for 4 days with 0, 0.1, or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases, and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS.EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features), and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine, 5'-methylthioadenosine, and the tryptophan metabolites, L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases.EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors, and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development.

Background This paper focuses on the relationships between health ‘policy’ as it is embodied in official documentation, and health ‘practice’ as reported and reflected on in the talk of policy-makers, health professionals and patients. The specific context for the study involves a comparison of policies relating to the secondary prevention of coronary heart disease (CHD) in the two jurisdictions of Ireland – involving as they do a predominantly state funded (National Health Service) system in the north and a mixed health-care economy in the south. The key question is to determine how the detail of health policy as contained in policy documents connects to and gets translated into practice and action. Methods The data sources for the study include relevant health-care policy documents (N = 5) and progress reports (N = 6) in the two Irish jurisdictions, and semi-structured interviews with a range of policy-makers (N = 28), practice nurses (14), general practitioners (12) and patients (13) to explore their awareness of the documents’ contents and how they saw the impact of ‘policy’ on primary care practice. Results The findings suggest that although strategic policy documents can be useful for highlighting and channelling attention to health issues that require concerted action, they have little impact on what either professionals or lay people do. Conclusion To influence the latter and to encourage a systematic approach to the delivery of health care it seems likely that contractual arrangements – specifying tasks to be undertaken and methods for monitoring and reporting on activity – are required.

Fetal alcohol syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates G-protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol's calcium transient and effect neural crest death are unknown. Here we show that ethanol's repression of the transcriptional effector β-catenin causes the neural crest losses. Clinically relevant ethanol concentrations (22-78 mM) rapidly deplete nuclear β-catenin from neural crest progenitors, with accompanying losses of β-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion, and survival. Using forced expression studies, we show that β-catenin loss of function (via dominant-negative T cell transcription factor [TCF]) recapitulates ethanol's effects on neural crest apoptosis, whereas β-catenin gain-of-function in ethanol's presence preserves neural crest survival. Blockade of ethanol's calcium transient using Bapta-AM normalizes β-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize β-catenin. We propose that ethanol's repression of β-catenin causes the neural crest losses in this model of FAS. β-Catenin is a novel target for ethanol's teratogenicity. β-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.

Prenatal ethanol exposure causes significant neurodevelopmental deficits through its induction of apoptosis in neuronal progenitors including the neural crest. Using an established chick embryo model, we previously showed that clinically relevant ethanol concentrations cause neural crest apoptosis through mobilization of an intracellular calcium transient. How the calcium transient initiates this cell death is unknown. In this study, we identify CaMKII as the calcium target responsible for ethanol-induced apoptosis. Immunostaining revealed selective enrichment of activated phosphoCaMKII(Thr286) within ethanol-treated neural crest. CaMKII activation in response to ethanol was rapid (< 60 s) and robust, and CaMKII activity was increased 300% over control levels. Treatment with CaMKII-selective inhibitors but not those directed against CaMKIV or PKC completely prevented the cell death. Forced expression of dominant-negative CaMKII prevented ethanol's activation of CaMKII and prevented the ethanol-induced death, whereas constitutively active CaMKII in ethanol's absence significantly increased cell death to levels caused by ethanol treatment. In summary, CaMKII is the key signal that converts the ethanol-induced, short-lived Ca(i) (2+) transient into a long-lived cellular effector. This is the first identification of CaMKII as a critical mediator of ethanol-induced cell death. Because neural crest differentiates into several neuronal lineages, our findings offer novel insights into how ethanol disrupts early neurogenesis.

Objective To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA). Methods OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN. Results Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P < 0.01) and tibial cartilage erosion increased with time (P < 0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P < 0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P < 0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P < 0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints. Conclusion Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

During the process of endochondral bone formation, the maturing chondrocyte exhibits profound changes in energy metabolism. To explore the mechanism of energy conservation in cartilage we examined the expression of creatine kinase, an enzyme that catalyzes the formation of ATP in tissues under oxygen stress. Measurement of creatine kinase activity and cytochemical assessment of enzyme distribution clearly showed that the level of enzyme activity was related to chondrocyte maturation. Thus, as the cells hypertrophied, there was a progressive increase in creatine kinase activity. Similarly, an elevation in creatine kinase activity was noted in chondrocyte cultures as the cells assumed an hypertrophic state. When cartilage calcification was disturbed by rickets, there was a decrease in enzyme activity in the hypertrophic region. Studies were performed to examine the creatine kinase isozyme profile of cells of the epiphysis. In resting and proliferating cartilage, the isoform was MM. In hypertrophic cartilage, the predominant isoforms were MB and BB. In terms of the creatine phosphate content, the highest values were seen in the proliferative region; lower amounts were present in hypertrophic and resting cartilage; and no creatine phosphate was detected in calcified cartilage. These data suggest that turnover of creatine phosphate is greatest in the mineralized region of the epiphysis. The results of these investigations point to creatine kinase as being under developmental control. The activity of the enzyme in cartilage cells should serve as a marker of developmental events associated with chondrocyte proliferation, hypertrophy, and mineralization.

Background Acute cough due to upper respiratory tract infection (URTI) is a common symptom. Non-prescription over-the-counter (OTC) medicines are frequently recommended as a first-line treatment, but there is little evidence as to whether these drugs are effective. Objectives To assess the effects of oral OTC cough preparations for acute cough. Search strategy We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2006, issue 4); MEDLINE (January 1966 to January Week 1, 2007); EMBASE (January 1974 to January 2007); and the UK Department of Health National Research Register (June 2007). Selection criteria Randomised controlled trials (RCTs) comparing oral OTC cough preparations with placebo in children and adults suffering from acute cough in ambulatory settings. We considered all cough outcomes and second outcomes of interest were adverse effects. Data collection and analysis Two review authors independently screened potentially relevant citations and independently extracted data and assessed study quality. Quantitative analysis was performed where appropriate. Main results Twenty five trials (17 in adults, 8 in children) involving 3492 people (2876 adults and 616 children) were included. Results of studies in adults: Six trials compared antitussives with placebo and had variable results. Two trials compared the expectorant, guaifenesin with placebo, one indicated significant benefit whereas the other did not. One trial found that a mucolytic reduced cough frequency and symptom scores. Two studies examined antihistamine-decongestant combinations and found conflicting results. Three studies compared other combinations of drugs with placebo and indicated some benefit in reducing cough symptoms. Three trials found antihistamines were no more effective than placebo in relieving cough symptoms. Results of studies in children: Antitussives (two studies), antihistamines (two studies), antihistamine decongestants (two studies) and antitussive/bronchodilator combinations (one study) were no more effective than placebo. No studies using expectorants met our inclusion criteria. The results of one trial favoured active treatment with mucolytics over placebo. One trial tested two paediatric cough syrups and both preparations showed a 'satisfactory response' in 46% and 56% of children compared to 21% of children in the placebo group. Authors' conclusions There is no good evidence for or against the effectiveness of OTC medicines in acute cough. The results of this review have to be interpreted with caution due to differences in study characteristics and quality. Studies often showed conflicting results with uncertainty regarding clinical relevance. Higher quality evidence is needed to determine the effectiveness of self-care treatments for acute cough. Plain language summary The evidence for effectiveness of oral over-the-counter cough medicines is weak Acute cough is a common and troublesome symptom in people who suffer from acute upper respiratory tract infection (URTI). Many people self-prescribe over-the-counter (OTC) cough preparations and health practitioners often recommend their use for the initial treatment of cough. The results of this review suggest that there is no good evidence for or against the effectiveness of OTC medications in acute cough. The results of this review have to be interpreted with caution because the number of studies in each category of cough preparations was small. Many studies were of low quality and very different from each other, making evaluation of overall efficacy difficult.

This is a summary, including tables of key findings and quality of included trials of a Cochrane review, published in this issue of EBCH, first published as: Smith SM, Schroeder K, Fahey T. Over-the-counter medications for acute cough in children and adults in ambulatory settings (Review). Cochrane Database of Systematic Reviews 2008, Issue No. 1. Art No.: CD001831. DOI:10.1.002/14 651 858.CD001831.pub3. Further information for this Cochrane review is available in this issue of EBCH in the accompanying Commentary. Copyright © 2009 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd. The Cochrane Collaboration

Objective To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model. Methods A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase–polymerase chain reaction. Results Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased. Conclusion The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Background: Prenatal ethanol exposure is a leading cause of mental retardation. Alcohol damages susceptible neuronal populations through its alteration of signaling pathways that direct cellular activity and survival. In early neural crest cells, ethanol elicits an intracellular Ca2+ transient that is necessary and sufficient to cause apoptosis. We tested the hypothesis that ethanol's activity represents a saturable and selective effect of alcohols upon this pathway. Methods: Fura-2–loaded chick embryos, at the 3-somite stage, were exposed to n-alcohols ranging in size from ethanol (C2) to decanol (C10). Thereafter, Ca2+ mobilization was measured using Fura-2 and ratiometric imaging. Apoptosis was assessed using acridine orange uptake. Results: Ethanol caused the dose-dependent mobilization of intracellular Ca2+ within neural crest populations, with an EC50 of 52.0 mM. n-Alcohols displayed increasing potency for Ca2+ mobilization through pentanol. Hexanol and heptanol were inactive. Unexpectedly, micromolar n-octanol concentrations triggered significant Ca2+ release and apoptosis in a G-protein–dependent manner. Decanol was inactive. Coaddition of either octanol or decanol antagonized the ability of ethanol to stimulate Ca2+ release. Conclusions: The selective, saturable effect of n-alcohols upon Ca2+ mobilization in neural crest is consistent with a hypothesis that ethanol stimulates these signals through specific interaction with one or more alcohol-binding sites on a target protein. Octanol may overcome structural constraints imposed upon C6 and C7 in interacting with this protein target; alternatively, it may interact through a unique binding site.

BACKGROUND Teratology studies must be carefully designed to minimize potential secondary effects of vehicle and delivery routes. A systematic method to evaluate chick models of early embryogenesis is lacking. METHODS We investigated 3 experimental approaches that are popular for studies of early avian development, in terms of their utility for teratogen assessment starting at gastrulation. These included in vitro embryo culture, egg windowing followed by direct application of a carrier vehicle to the embryo, and injection of a carrier vehicle into the egg yolk. We also developed a morphologically based scoring system to assess development of the early embryo. RESULTS The in vitro culture and egg windowing approaches both caused an unacceptably high incidence of central nervous system and cardiac abnormalities in vehicle-treated embryos, which made it difficult to identify teratogen-specific defects. In contrast, exposing chick embryos to vehicle via direct egg yolk injection did not induce developmental anomalies. CONCLUSIONS Optimization of the exposure route of potential toxicants to the embryo is critical because control treatments can cause developmental anomalies. In ovo yolk injection minimizes perturbation of young embryos and may be appropriate for teratogen delivery. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.

Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient.Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes.Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells.We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.

Retinoic acid (RA) is an essential mediator of embryogenesis. Some, but not all, of its targets have been identified. We previously developed a rat model of gestational retinoid deficiency (RAD; Power et al. [1999] Dev. Dyn. 216:469–480) and generated embryos with developmental impairments that closely resemble genetic and dietary models of retinoid insufficiency. Here, we used microarray analysis and expression profiling to identify 88 transcripts whose abundance was altered under conditions of retinoid insufficiency, as compared with normal embryos. Among these, the induction by RAD of genes involved in collagen I synthesis (COL1A1, IA2 and VA2, prolyl-4-hydroxylase-α1) and protein galactosylation (galactokinase, ABO galactosyltransferase, UDP-galactose transporter-related protein) was especially noteworthy because extracellular matrix regulates many developmental events. We also identified several genes involved with stress responses (cathepsin H, UBC2E, IGFBP3, smoothelin). Real-time polymerase chain reaction analysis of selected candidates revealed excellent agreement with the array findings. Further validation came from the demonstration that these genes were similarly dysregulated in two genetic models of retinoid insufficiency, the retinol binding protein null-mutant embryo and the Raldh2 null-mutant embryo. In situ hybridization of RAD embryos found increased collagen IA1 and IGFBP3 mRNA within the connective mesenchyme and vasculature, respectively, and a failure to repress the growth factor midkine within the RAD neural tube. Many of the identified genes were not known previously to respond to retinoid status and will provide new insights to retinoid roles and to the consequences of retinoid insufficiency. Developmental Dynamics 229:886–898, 2004. © 2004 Wiley-Liss, Inc.

Objectives To assess the prevalence of undiagnosed diabetes, impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) in patients over the age of 40 years attending their general practitioner (GP) in Ireland, through opportunistic screening, using a three-step screening tool involving self-determined high-risk groups, random venous plasma glucose (RVPG) measurement and oral glucose tolerance tests. Design In participating general practices, 100 consecutive patients > 40 years, completed a screening questionnaire relating to diabetes-related symptoms and risk factors. Patients with previously diagnosed diabetes were not excluded from the study and the screening instrument included a question about known diabetes. Patients without known diabetes mellitus (DM) and with at least two risk factors and/or symptoms underwent a RVPG test. Those with an RVPG above 5.5 mmol/l underwent an oral glucose tolerance test. Results Forty-one practices returned 3821 questionnaires. The prevalence of Type 2 diabetes mellitus in the study population was 9.2% (353), of whom 23.5% (83) were previously undiagnosed. DM was detected on the basis of an RVPG > 11.1 mmol/l in 0.8% (32) of the studied population. DM was detected on the basis of the oral glucose tolerance test in 1.3% (51) of the population. One per cent (39) had a fasting plasma glucose (FPG) ≥ 7.0 mmol/l, 0.6% (24) had a 2-h > 11.0 mmol/l and 0.3% (12) had both. Diabetes would not have been detected in 12 people had the 2-h test been omitted. The prevalence rate for IFG and/or IGT was 3.9% (148). Of the 103 patients with IGT, 83 (81%) would have been missed had the GTT been omitted. Conclusion Opportunistic diabetes screening in general practice using a screening questionnaire followed by RVPG testing and GTT for those above 5.5 mmol/l is feasible, with a high participation rate. The use of GTTs rather than fasting glucose testing alone improves patient identification, in particular those with IGT who are at higher cardiovascular risk.

Background: Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis. Methods: Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange. Results: Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis. Conclusions: Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.

The functional diversity of adenylyl cyclases provides for different modes of cyclic AMP signalling in mammals. This study reports the cloning and functional characterisation of a cDNA encoding human adenylyl cyclase IX (ACIX). The data show that human ACIX is a Ca2+/calcineurin-inhibited adenylyl cyclase prominently expressed in vital organs, including brain, heart, and pancreas. ACIX mRNA was detected in several brain regions, including neocortex, hippocampus, striatum, and cerebellum. By in situ hybridisation, ACIX mRNA was localised to pyramidal and granule cells of the hippocampus, indicating that it is expressed predominantly in nerve cells. Further analysis of ACIX mRNA expression revealed two major forms of ACIX mRNA that arose through tissue-specific differential mRNA polyadenylation. Taken together, the data show that (a) human ACIX is under inhibitory control by Ca2+ through calcineurin, (b) ACIX may be involved in higher brain functions, and (c) post-transcriptional regulation of ACIX gene expression is a species-specific control mechanism that may enhance the versatility of cyclic AMP signalling in humans.

Background: Understanding the basis for ethanol's teratogenic effects may inform the etiology of fetal alcohol syndrome. Here we investigate how genetic background and susceptibility to ethanol-induced neural crest apoptosis contribute to the distinctive craniofacial phenotype observed after prenatal alcohol exposure. Methods: Nine different chick strains were exposed to ethanol at gastrulation. The sensitivity of these embryos to ethanol-induced neural crest apoptosis was reported elsewhere (Debelak and Smith, 2000). Here, these embryos were permitted to develop until embryonic day 10, when facial morphogenesis was largely complete, and cephalometric measurements were made on cleared skulls. Shifts in facial growth were correlated against the severity of ethanol-induced apoptosis in facial precursors. Results: The facial shape produced by ethanol exposure was a function of the embryos' genetic strain. Three general responses were observed: apparent midfacial flattening (Babcock B300 x Hampshire Red, ISA-Babcock, HyLine W98, and HyLine W36 strains), overall facial expansion (Spafas and Babcock B300 strains), or overall facial hypoplasia (DeKalb strains). When dose and timing of exposure were held constant, the embryo's genetic background predicted the facial outcome. For ethanol-sensitive strains, apoptosis of facial precursor populations was required to produce the facial defects. That some strains had essentially normal faces despite extensive cell death indicated a capacity to recover from the earlier neural crest losses. Conclusions: We propose that ethanol's effects on craniofacial development are multifactoral, and these influences may include susceptibility to apoptosis, regenerative capacity, and compensatory outgrowth of the facial primordia. The embryo's genetic background may modulate these events. The high and low responder chick strains are useful tools to dissect these contributions.

Background: Ethanol-induced neural crest apoptosis likely contributes to the distinctive craniofacial phenotype that results from prenatal alcohol exposure. The mechanism responsible for this apoptosis is incompletely understood. A serendipitous change in poultry production flocks led to the discovery that, in chick, the embryo's genetic background modulates its susceptibility to ethanol-induced apoptosis. Methods: We examined the level of ethanol-induced neural crest apoptosis in 11 chick layer strains or crosses, using acridine orange uptake. Results: Holding the ethanol dose and exposure stage constant, strains were classified into very sensitive (Babcock ISA, HyLine W98, Babcock B300/Hampshire Rcd cross [BxHR]), moderately sensitive (Spafas, HyLine W36, Babcock B300), and nonresponsive (DeKalb White and Black, Shaver White and 2000, DeKalb White/Hampshire Red cross). Detailed examination of two susceptible strains (W98, BxHR) and a resistant strain (DeKalb White) revealed that the DeKalh's non response was not caused by a shift in timing of apoptosis, or to a lower alcohol exposure at cither time of injection or time of death. Strains had identical stage distributions at the time of injection and at apoptosis; housing and diet were held constant. Conclusions: Factors within the embryo and/or egg environment can affect the susceptibility to ethanol-induced apoptosis. These sensitive and resistant strains will be important tools to dissect the molecular mechanism of ethanol-induced apoptosis, and for understanding how these losses affect subsequent development.

Background: Increasing evidence demonstrates that genetic background is an important modulator of alcohol's effects on the developing fetus. Such effects are separable from maternal ethanol metabolism. Here, we study ethanol's effects on cardiogenesis in an avian model that shows strong cell death within neuronal and neural crest precursors following ethanol exposure. Methods: The study design tested the hypothesis that ethanol-induced losses of cardiac neural crest populations would disrupt outflow tract development and thus contribute to the valvuloseptal deficits observed in prenatal alcohol exposure. Three chick strains were exposed to alcohol at gestational windows between gastrulation and early heart septation (day 3 incubation), and then hearts were examined at the completion of morphogenesis (day 10 incubation). Results: Ethanol's impact on cardiac development was influenced by fetal genetics. The B300 X Hampshire Red cross exhibited pronounced cell death within cardiac neural crest populations but had normal development of the heart and aortic arches. Neural crest migration and differentiation into the distal outflow tract were also normal in these embryos, which suggested a capacity to repair earlier losses. The DeKalb White X Hampshire Red cross also did not exhibit cardiac defects. Hearts of the B300 strain had a unique phenotype with respect to ethanol exposure and exhibited a thin ventricular compact layer, dilatation, and reduced myosin/deoxyribonucleic acid and myosin/protein content, a phenotype that indicates disrupted myocardial maturation and inductive cues. The deficit was only observed when ethanol exposure occurred at stages 15 or 18 and apparently was independent of neural crest cell death. Such ventricular thinning might go undetected in the absence of extensive screening. Conclusions: Results add to the increasing evidence that genetic background strongly modulates the effects of prenatal alcohol exposure. The results also suggest that embryos have a varying capacity to repair and recover from earlier neural crest losses.

Retinoids long have been implicated in limb development and their endogenous contributions to this process are finally being elucidated. Here we use an established model of retinoid depletion during specific gestational windows to investigate the role of endogenous retinoic acid (RA) in supporting limb outgrowth. Rat embryos were deprived of RA starting at days-postcoitum (dpc) 3.0, 5.5, or 7.0 and harvested at the 35-somite stage (dpc 12–12.5). Although embryos from all these windows possessed many characteristics of gestational retinoid deficiency (frontonasal hypoplasia, straight tail, reduced CRBPI and RARβ), their limb buds emerged with only modest size reductions. Molecular analysis of RA-deficient limb buds revealed enhanced gli-3 and reduced hoxd-12, hoxd-13, shh, and fgf-4, while fgf-8, en-1, and wnt-7a expression remained unaltered. Occasional posterior truncations were observed at low incidence in the longest deficiency window; otherwise, the deficiency window length had no discernable impact on the severity of these changes. At the 45-somite stage, RA-deficient limbs had additional losses of hoxd-13 and fgf-8, accompanied by a flattened AER, suggestive of an ultimate failure in limb bud outgrowth. Results could not confirm a function for endogenous retionids in limb initiation, but show they are required to maintain the signaling loops between the developing mesenchyme and AER that govern limb outgrowth after the initial emergence of limb bud. Dev Dyn 1999;216:469–480. ©1999 Wiley-Liss, Inc.

Fibrous dysplasia (FD) is a progressive bone disease in which abnormal fibroblast proliferation results in the replacement of normal cancellous bone with an immature fibrous tissue that is poorly mineralized. The disease manifests itself in the monostotic form in which only one bone is involved and the polyostotic form in which multiple bones at different sites are affected. The McCune-Albright syndrome is a variation of the polyostotic form in which patients demonstrate a greater extent of bone involvement and a variety of endocrinopathies. Somatic activating mutations in the GNAS gene have been demonstrated in the fibrotic lesions of patients affected with either monostotic or polyostotic FD. The increased cAMP levels caused by the G-protein mutations lead to increased interleukin-6 (IL-6) levels in the affected tissues, resulting in abnormal osteoblast differentiation and increased osteoclastic activity. Utilizing cell culture techniques that have been developed for mammalian bone marrow stromal cells, we have successfully cultured osteogenic stem cells from the affected stroma of 11 FD patients. Cells cultured from patients with polyostotic FD showed a high frequency of the Gsalpha mutation, whereas cells from monostotic FD patients showed a low frequency of the mutation. Both the normal and FD cells displayed the osteogenic phenotype when exposed to medium containing glucocorticoids. Glucocorticoids also caused a dramatic inhibition of IL-6 mRNA and protein levels in osteogenic cells cultured from the FD patients. These findings suggest that chemical alteration of cellular function may lead to new treatment options for patients with FD.

The use of hydrogenated rapeseed oil as a carrier for conidia of the entomopathogenic fungus Beauveria bassiana (Bals) Vuill was investigated as part of a research programme on the control of the larger grain borer, Prostephanus truncatus (Horn). Melting the oil, which is solid at temperatures below 32 °C, allows the incorporation of materials such as aggregation pheromones and conidia; sudden cooling produces solid fat pellets. In attraction tests conducted with pellets containing P truncatus aggregation pheromone, significantly higher numbers of beetles were attracted to pellets containing pheromone at a concentration of 4 ml litre−1 compared to control pellets for at least four weeks when held in Petri dishes in the laboratory and for at least six weeks when pellets held in insect traps were exposed to outside conditions. The attraction was retained over a period of storage in glass bottles; pellets stored in the freezer or incubator (at −10 °C or 27 °C) attracted beetles according to the pheromone level for the duration of the work (14 and 13 months respectively). When pheromones and conidia were incorporated into the same pellets they could be stored in a freezer or refrigerator retaining over 80% viability after 51 weeks; those stored in an incubator at 27 °C showed significantly lower germination at 20.7–27.2% after the same time. There was an indication that the pheromone caused a slight reduction in the viability of conidia, although this may have been just a slight delay in the speed of germination. Rapid dose transfer from pellets with conidia with and without the addition of pheromone was demonstrated. Insects were exposed to pellets for 24 hours and 96–100% mortality was observed in treatments containing conidia within six days of exposure. © 1999 Society of Chemical Industry

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardiac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough [1994] Dev. Dyn. 200:155-162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell explants. Fluorography of proteins from endoderm-conditioned medium revealed 1-2 dozen bands, the most prominent of which migrated at approximately 17 and 25 kD. The bulk of the 17-kD band, which migrates near FGFs and subunits of the transforming growth factor-beta family, was identified by N-terminal sequencing as transthyretin (TTR). A component of the 25-kD band was identified by Western blotting as retinol binding protein (RBP). RT/PCR analysis revealed that mRNAs for both proteins are in the embryo as early as stage 3. In situ hybridization localized these mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, RBP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of RBP by stage 7, followed by sequential increases to stage 25; by contrast, content of RBP in isolated hearts peaked at stage 14, then declined. Immunohistochemistry revealed the presence of RBP protein in the extracellular matrix subjacent to lateral plate endoderm beginning at stage 8; upon formation of the definitive heart, intense staining was observed in the cardiac "jelly." By contrast TTR was intracellular, first detected as subtle deposits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanchnic mesoderm. At stages 11-14, TTR was detected only in myocardial cells. Such localization of RBP and TTR may indicate a role in the transport and distribution of retinol and thyroid hormone, respectively, from yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development.

The ability of both acute and chronic ethanol exposures to elicit cell death within specific embryonic and adult tissues is believed to partly underlie ethanol's pathogenicity; however, the mechanism underlying this cell death is unknown. This study partially characterized the mechanism of ethanol-induced neural crest cell death in a chick embryo model of fetal alcohol syndrome. In situ DNA end-labeling demonstrated this cell death was apoptotic and occurred at embryonic ethanol levels as low as 42 mM. Regardless of the initial exposure time, this apoptosis always appeared at a distinct developmental time point simultaneous with the normal deletion of a cranial neural crest subset. This suggested that ethanol might act through aberrant activation of the endogenous death pathway; however, ethanol exposure failed to induce two components of this pathway, the homeotic transcription factor msx-2 and the growth factor bone morphogenetic protein 4. Both endogenous and ethanol-induced death were blocked by local application of an interleukin-1β converting enzyme/CED-3 protease (caspase) inhibitor, showing that the two paths converge mechanistically and suggesting the potential to prevent this aspect of ethanol's teratogenicity. Ethanol exposure did not significantly alter cell proliferation within neural crest-populated regions, suggesting that susceptibility to ethanol-induced death did not involve exit from the cell cycle. Apoptotic deletion of cranial neural crest could partially explain the craniofacial deficits characteristic of the fetal alcohol syndrome.

Early heart development is known to be sensitive to retinoid concentrations; a specific pattern of malformations is observed in both vitamin A-deficiency and retinoid-toxicity states. While the influence of retinoids on early cardiac morphogenesis has been described previously, the effect of retinoids upon cardiomyocyte differentiation and gene expression is largely uncharacterized. We have established an in ovo chick embryo model in which slow-release retinoic acid (RA) induces four distinct cardiac malformations in a dose-dependent fashion. Late primitive streak-stage chick embryos were treated with all-trans-retinoic acid released from anion exchange beads placed on the embryo's left side and then allowed to develop further for 20-24 hr. At low doses (10 and 25 micrograms/ml RA) an abnormal loop structure was observed. At higher doses (50 and 100 micrograms/ml RA) cardia bifida and clustered heart tissue were noted. Situs inversus only occurred after treatment with 100 micrograms/ml RA. RA-treated embryos were subsequently analyzed for appropriate cardiac myocyte differentiation using antibody staining and ELISA analysis to detect sarcomeric myosin heavy chain, tropomyosin, titin, and alpha-actinin protein expression. Alpha-actinin expression was significantly decreased in RA-treated embryos, as compared to DMSO-treated controls. Also, heart contraction rate was depressed after RA exposure. RA exposure did not alter the protein expression levels of sarcomeric myosin heavy chain or tropomyosin. The observed alterations are consistent with suggestions that retinoids may affect both morphogenesis and myofibril formation in the developing heart.

Fetal alcohol syndrome (FAS) is characterized by growth retardation, mental deficiencies, and numerous craniofacial and neuronal anomalies; the type and severity of these defects may be related to the time and dose of maternal ethanol exposure. Ethanol administered during presomitic stages results in the typical FAS craniofacial phenotype and is accompanied by a loss of cranial neural crest cells (CNCCs) through ethanol-induced cell death. However, the stage-specific effects of ethanol on the CNCC population is unknown. We examined the effects of ethanol on CNCC populations by treating in ovo chick embryos with a single ethanol dose (0.43 mmol/egg) at various stages of CNCC development, and corresponding to the first 3-4 weeks of human gestation. Ethanol treatment induced cell death and reduced CNCC populations in patterns consistent with observed dysmorphologies of CNCC-derived cranial structures. The precise population affected was dependent on the timing of ethanol exposure. Treatment at gastrulation or neurulation induced cell death and losses of CNCC populations, particularly those in rostral positions, and resulted in more severe craniofacial defects. In contrast, treatment at early somitic stages (4-16 somites) induced cell death, primarily within caudal CNCC populations, but resulted in less severe craniofacial defects, suggesting an increased capacity for recovery. These results suggest that there are distinct developmental windows during which the CNCCs may be particularly susceptible to ethanol-induced cell death. We conclude that ethanol exposure seems to affect specific events adversely during neural crest development. The timing of embryonic ethanol exposure relative to CNCC development could account, in part, for the heterogenous craniofacial defects observed in FAS.

Fetal alcohol syndrome (FAS) is characterized by growth retardation, craniofacial malformations, and heart and neural defects; the cellular and molecular mechanism(s) responsible for ethanol's teratogenicity remains unknown. Although the phenotype suggests that prenatal ethanol exposure perturbs neural crest cell development, direct proof that these cells are an in utero target is still lacking. Previous research suggested that cranial neural crest cells are eliminated by ethanol-induced apoptosis. We tested this hypothesis using a chick embryo model of FAS. A single dose of ethanol, chosen to achieve a concentration of 35-42 mg/dl, was injected in ovo at gastrulation and resulted in growth retardation, craniofacial foreshortening, and disrupted hindbrain segmentation. Ethanol exposure enhanced cell death within areas populated by cranial neural crest cells, particularly in the hindbrain and craniofacial mesenchyme. In contrast, control embryos had limited cell death within these regions. Subsequent immunolabeling with neural crest cell-specific antibody revealed that ethanol treatment resulted in fewer neural crest cell numbers, whereas neural crest migration patterns were unaffected by ethanol. These results suggest that prenatal ethanol exposure leads to loss of cranial neural crest cells. Such a loss could result, in part, in the phenotype characteristic of FAS.