Understanding the importance of low-molecular weight (ethylene oxide- and propylene oxide-induced) DNA adducts and mutations in risk assessment: Insights from 15 years of research and collaborative discussions.

The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An extended effort to identify key questions and collect relevant data to address them was focused on the ubiquitous low MW N7-alkyl/hydroxyalkylguanine adducts. Several academic, governmental, and industrial laboratories collaborated to gather new data aimed at better understanding the role and potential impact of these adducts in quantifiable genotoxic events (gene mutations/micronucleus). This review summarizes and evaluates the status of dose-response data for DNA adducts and mutations from recent experimental work with standard mutagenic agents and ethylene oxide and propylene oxide, and the importance for risk assessment. This body of evidence demonstrates that small N7-alkyl/hydroxyalkylguanine adducts are not pro-mutagenic and, therefore, adduct formation alone is not adequate evidence to support a mutagenic mode of action. Quantitative methods for dose-response analysis and derivation of thresholds, benchmark dose (BMD), or other points-of-departure (POD) for genotoxic events are now available. Integration of such analyses of genetox data is necessary to properly assess any role for DNA adducts in risk assessment. Regulatory acceptance and application of these insights remain key challenges that only the regulatory community can address by applying the many learnings from recent research. The necessary tools, such as BMDs and PODs, and the example datasets, are now available and sufficiently mature for use by the regulatory community. Environ. Mol. Mutagen. 60: 100-121, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

CHD1 loss sensitizes prostate cancer to DNA damaging therapy by promoting error-prone double-strand break repair.

BACKGROUND: Deletion of the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1) is a common genomic alteration found in human prostate cancers (PCas). CHD1 loss represents a distinct PCa subtype characterized by SPOP mutation and higher genomic instability. However, the role of CHD1 in PCa development in vivo and its clinical utility remain unclear. PATIENTS AND METHODS: To study the role of CHD1 in PCa development and its loss in clinical management, we generated a genetically engineered mouse model with prostate-specific deletion of murine Chd1 as well as isogenic CHD1 wild-type and homozygous deleted human benign and PCa lines. We also developed patient-derived organoid cultures and screened patients with metastatic PCa for CHD1 loss. RESULTS: We demonstrate that CHD1 loss sensitizes cells to DNA damage and causes a synthetic lethal response to DNA damaging therapy in vitro, in vivo, ex vivo, in patient-derived organoid cultures and in a patient with metastatic PCa. Mechanistically, CHD1 regulates 53BP1 stability and CHD1 loss leads to decreased error-free homologous recombination (HR) repair, which is compensated by increased error-prone non-homologous end joining (NHEJ) repair for DNA double-strand break (DSB) repair. CONCLUSIONS: Our study provides the first in vivo and in patient evidence supporting the role of CHD1 in DSB repair and in response to DNA damaging therapy. We uncover mechanistic insights that CHD1 modulates the choice between HR and NHEJ DSB repair and suggest that CHD1 loss may contribute to the genomic instability seen in this subset of PCas.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

Elevated tissue factor expression contributes to exacerbated diabetic nephropathy in mice lacking eNOS fed a high fat diet.

BACKGROUND: Human eNOS (NOS3) polymorphisms that lower its expression are associated with advanced diabetic nephropathy (DN), and the lack of eNOS accelerates DN in diabetic mice. Diabetes is associated with fibrin deposition. Lack of nitric oxide and fatty acids stimulates the NF-kB pathway, which increases tissue factor (TF). OBJECTIVES: To test the hypothesis that TF contributes to the severity of DN in the diabetic eNOS(-/-) mice fed a high-fat diet (HF). METHODS: We made eNOS(-/-) and wild-type mice diabetic with streptozotocin. Half of them were placed on HF. RESULTS: Blood glucose levels were not affected by either the diet or eNOS genotype. Lack of eNOS in the diabetic mice increased urinary albumin excretion, glomerulosclerosis, interstitial fibrosis, and glomerular basement membrane thickness. HF by itself did not affect DN in the wild-type mice, but significantly enhanced DN in eNOS(-/-) mice. More than half of diabetic eNOS(-/-) mice on HF died prematurely with signs of thrombotic complications. Diabetic kidneys contained fibrin and TF, and their levels were increased by the lack of eNOS and by HF in an additive fashion. The HF diet increased the kidney expression of inflammatory genes. The increase in TF preceded DN, and administration of an anti-mouse TF antibody to diabetic mice reduced the expression of inflammatory genes. CONCLUSION: Together, these data indicate a causal link between TF and the exacerbation of DN in eNOS(-/-) mice. The condition is significantly worsened by enhanced inflammatory responses to an HF diet via TF.

A putative 'pre-nervous' endocannabinoid system in early echinoderm development.

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

[Identification of VHL-associated changes in clear cell renal carcinoma: the application of combined genome and expression analyses]. / Identifizierung VHL-assoziierter Veränderungen im klarzelligen Nierenzellkarzinom: Anwendung von kombinierten Genom- und Expressionsanalysen.

Sporadic renal cell carcinoma (RCC) represents a heterogeneous tumor, which is traditionally classified into subtypes based on morphological criteria. In recent years high-throughput molecular analyses have been able to identify genomic and proteomic alterations in tumor cells. These markers are the basis for a molecular classification of RCC and bear prognostic value. However, an isolated consideration of genomic and proteomic alterations prevents deeper insights into the complex processes of carcinogenesis. Here we summarize recent studies focussing on this aspect of RCC and present a systems biology concept for the identification of novel tumor markers. These could be applied to improve future diagnosis and therapy of RCC.

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response.

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1alpha. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

Thrombolytic treatment for stroke in the Scandinavian countries.

OBJECTIVE: We wanted to describe the use of thrombolytic treatment for stroke in Scandinavia, to assess stroke doctors' opinions on this treatment, to identify barriers against treatment, and to suggest improvements to overcome these barriers. METHODS: We sent questionnaires to 493 Scandinavian doctors, who were involved in acute stroke care. RESULTS: We received 453 (92%) completed questionnaires. Overall, 1.9% (range per hospital 0-13.9%) of patients received thrombolytic treatment. A majority (94%) of the respondents was convinced of the beneficial effects of thrombolytic treatment and many (85%) felt that its risks were acceptable. Main barriers were: unawareness of stroke symptoms among patients (82%) and their failure to respond adequately (54%); ambulance services not triaging acute stroke as urgent (23%); and insufficient in-hospital routines (15%). The respondents suggested that the following measures should be prioritized to increase the treatment's use: educational programmes to improve public awareness on stroke and how to respond (96%); education of in-hospital (88%) and prehospital (76%) medical staff. CONCLUSIONS: A large majority of Scandinavian doctors regard thrombolytic treatment for stroke as beneficial, yet its implementation in clinical practice has so far been poor. Our survey identified important barriers and potential measures that could increase its future use.

Prestroke physical activity is associated with severity and long-term outcome from first-ever stroke.

OBJECTIVE: To determine whether prestroke level of physical activity influenced stroke severity and long-term outcome. METHODS: Patients included into the present analyses represent a subset of patients with first-ever stroke enrolled into the ExStroke Pilot Trial. Patients with ischemic stroke were randomized in the ExStroke Pilot Trial to an intervention of repeated instructions and encouragement to increase the level of physical activity or to a control group. Prestroke level of physical activity was assessed retrospectively by interview using the Physical Activity Scale for the Elderly (PASE) questionnaire. The PASE questionnaire quantifies the amount of physical activity done during a 7-day period. In this prospectively collected patient population initial stroke severity was measured using the Scandinavian Stroke Scale and long-term outcome was assessed after 2 years using the modified Rankin Scale. Statistical analyses were done using ordinal logistic regression. RESULTS: Data from 265 patients were included with a mean (SD) age of 68.2 (12.2) years. Confirming univariable analyses, multivariable analyses showed that patients with physical activity in the top quartile more likely presented with a less severe stroke, OR 2.54 (95% CI 1.30-4.95), and had a decreased likelihood of poor outcome, OR 0.46 (95% CI 0.22-0.96), compared to patients in the lowest quartile. CONCLUSIONS: In the present study physical activity prior to stroke was associated with a less severe stroke and better long-term outcome.

[Von-Hippel-Lindau gene mutation types. Association of gene expression signatures in clear cell renal cell carcinoma]. / Von-Hippel-Lindau-Gen-Mutationstypen. Assoziation mit Genexpressionssignaturen in klarzelligen Nierenzellkarzinomen.

AIMS: The von-Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may specifically alter pVHL functions, which have significant impact on gene expression and, consequently, on the disease outcome. The aim of this study was to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC. METHODS: Total RNA and genomic DNA were extracted from 94 frozen clear cell (ccRCC) and 21 papillary RCC (pRCC) specimens from the tumor biobank of Zurich University Hospital. Transcriptome analysis was performed using Affymetrix HG U133A gene chips. All ccRCC tumors were subjected to VHL gene mutation analysis. RESULTS: By applying significance analysis of microarrays genes were identified, which were differentially regulated among the tumor subgroups. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. A total of 186 differentially expressed genes were identified by comparing the gene expression profiles of ccRCC with VHL loss-of-function mutations and ccRCC with no gene alterations. CONCLUSIONS: The results clearly argue for a significant influence of VHL mutations on gene expression profiles in RCC.

Hereditary hemochromatosis genotypes and risk of ischemic stroke.

OBJECTIVE: We tested the hypothesis that the HFE genotypes H63D/H63D, H63D/wild type, C282Y/H63D, C282Y/C282Y, and C282Y/wild type are risk factors for symptomatic carotid atherosclerosis, ischemic cerebrovascular disease (ICVD), and ischemic stroke. METHODS: We performed an age- and gender-matched case-control study of 701 cases with symptomatic carotid atherosclerosis vs 2,777 controls, and a prospective study of 9,178 individuals from the Danish general population followed for 24 years, during which 504 developed ICVD, of whom 393 had ischemic stroke. RESULTS: Genotype was not consistently associated with symptomatic carotid atherosclerosis. The cumulative incidences of ICVD and ischemic stroke by age were increased for H63D/H63D vs wild type/wild type (log-rank: p = 0.003 and p < 0.001). H63D/H63D vs wild type/wild type had an age- and multifactorially adjusted hazard ratio of 2.0 (95% CI: 1.2 to 3.2; p = 0.007) and 2.1 (1.3 to 3.5; p = 0.004) for ICVD and of 2.4 (1.4 to 4.0; p = 0.001) and 2.8 (1.7 to 4.6; p < 0.001) for ischemic stroke; these remained significant after correction for multiple comparisons. Other hereditary hemochromatosis genotypes were not associated with ICVD or ischemic stroke. CONCLUSIONS: Hereditary hemochromatosis H63D homozygosity predicts a two- to threefold risk of ICVD and ischemic stroke.

Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate.

Endogenous creatinine clearance (Ccr) is widely accepted as an estimate of glomerular filtration rate (GFR), the best overall biomarker of kidney function. However, current common methods of measuring creatinine are not sensitive enough for mouse plasma. Accordingly, we here report a new method of measuring creatinine by liquid chromatography tandem mass spectrometry (LC-MS/MS) using deuterated [2H3]-creatinine as an internal standard. The assay requires 10 microl or less of plasma or urine, and is eight times more sensitive than high-performance liquid chromatography. The reproducibility of the assay of replicates is approximately +/-10%. The plasma creatinine levels of wild type male C57BL/6J mice obtained by LC-MS/MS are 0.076+/-0.002 mg/dl (n=65). To estimate daily urinary creatinine excretion for calculating Ccr, we collected urine from mice housed in metabolic cages, and combined this with washes from the cage internal surfaces. Creatinine in the wash varies from 4 to 67% of the total daily urinary creatinine excretion (typically approximately 400 microg/day). Ccr obtained by LC-MS/MS was 329+/-17 microl/min, which is indistinguishable from GFR measured by using fluorescein isothiocyanate-inulin. The LC-MS/MS method is sensitive, specific, simple, fast, and inexpensive; it is suitable for estimating GFR in conscious mice or other small animals. As it allows repeated measurements in the same animals, it facilitates detection of subtle differences or changes in renal function.

Stroke incidence and prevalence in Europe: a review of available data.

Reliable data on stroke incidence and prevalence are essential for calculating the burden of stroke and the planning of prevention and treatment of stroke patients. In the current study we have reviewed the published data from EU countries, Iceland, Norway, and Switzerland, and provide WHO estimates for stroke incidence and prevalence in these countries. Studies on stroke epidemiology published in peer-reviewed journals during the past 10 years were identified using Medline/PubMed searches, and reviewed using the structure of WHO's stroke component of the WHO InfoBase. WHO estimates for stroke incidence and prevalence for each country were calculated from routine mortality statistics. Rates from studies that met the 'ideal' criteria were compared with WHO's estimates. Forty-four incidence studies and 12 prevalence studies were identified. There were several methodological differences that hampered comparisons of data. WHO stroke estimates were in good agreement with results from 'ideal' stroke population studies. According to the WHO estimates the number of stroke events in these selected countries is likely to increase from 1.1 million per year in 2000 to more than 1.5 million per year in 2025 solely because of the demographic changes. Until better and more stroke studies are available, the WHO stroke estimates may provide the best data for understanding the stroke burden in countries where no stroke data currently exists. A standardized protocol for stroke surveillance is recommended.

D-amphetamine improves cognitive deficits and physical therapy promotes fine motor rehabilitation in a rat embolic stroke model.

BACKGROUND AND PURPOSE: The purpose of this study was to examine the effects of D-amphetamine (D-amph) and physical therapy separately or combined on fine motor performance, gross motor performance and cognition after middle cerebral artery thromboembolization in rats. METHODS: Seventy-four rats were trained in appropriate cognitive and motor behaviours. Thirteen animals were sham-operated and fifty-nine animals were embolized in the right carotid territory. Animals were randomly assigned to five groups: 1) SHAM (non-embolized, saline), 2) CONTROL (embolized, saline), 3) D-AMPH (embolized, D-amph), 4) THERAPY (embolized, saline + physical therapy) and 5) D-AMPH + THERAPY (embolized, D-amph + physical therapy). Rats of the groups 4-5 underwent d-amph or saline treatment on days 1, 3, 5 and 7 after surgery and were re-trained for 1 h starting 60 min after each treatment. During this time, rats were allowed to voluntarily engage in suitable cognitive or motor behaviours in order to obtain food. Animals from all groups were re-tested during days 21-28 after surgery. RESULTS: No differences in infarct volumes were observed between the groups of embolized animals. When evaluating performances on days 21-28 after surgery, rats of the SHAM and THERAPY groups had better fine motor performance than those of the CONTROL (P < 0.05), whereas rats of SHAM and D-AMPH groups achieved better cognitive performance than CONTROL rats (P < 0.05). No significant differences were observed between any groups regarding gross motor performance. CONCLUSIONS: After embolization, physical therapy improved fine motor performance and D-amph accelerated rehabilitation of cognitive performance as observed in the rats of the THERAPY and D-AMPH groups. As a result of the administration of a high dose of D-amph, the rats of the D-AMPH + THERAPY combination group failed to engage in physical therapy during D-amph intoxication, thereby limiting any promotion of rehabilitation by combining physical therapy and D-amph.

Significant variation in mortality and functional outcome after acute ischaemic stroke between Western countries: data from the tinzaparin in acute ischaemic stroke trial (TAIST).

BACKGROUND: The medical care of patients with acute stroke varies considerably between countries. This could lead to measurable differences in mortality and functional outcome. OBJECTIVE: To compare case mix, clinical management, and functional outcome in stroke between 11 countries. METHODS: All 1484 patients from 11 countries who were enrolled into the tinzaparin in acute ischaemic stroke trial (TAIST) were included in this substudy. Information collected prospectively on demographics, risk factors, clinical features, measures of service quality (for example, admission to a stroke unit), and outcome were assessed. Outcomes were adjusted for treatment assignment, case mix, and service relative to the British Isles. RESULTS: Differences in case mix (mostly minor) and clinical service (many of prognostic relevance) were present between the countries. Significant differences in outcome were present between the countries. When assessed by geographical region, death or dependency were lower in North America (odds ratio (OR) adjusted for treatment group only = 0.52 (95% confidence interval, 0.39 to 0.71) and north west Europe (OR = 0.54 (0.37 to 0.78)) relative to the British Isles; similar reductions were found when adjustments were made for 11 case mix variables and five service quality measures. Similarly, case fatality rates were lower in North America (OR = 0.44 (0.30 to 0.66)) and Scandinavia (OR = 0.50 (0.33 to 0.74)) relative to the British Isles, whether crude or adjusted for case mix and service quality. CONCLUSIONS: Both functional outcome and case fatality vary considerably between countries, even when adjusted for prognostic case mix variables and measures of good stroke care. Differing health care systems and the management of patients with acute stroke may contribute to these findings.

Is the effect of alcohol on risk of stroke confined to highly stressed persons?

BACKGROUND: Psychological stress and alcohol are both suggested as risk factors for stroke. Further, there appears to be a close relation between stress and alcohol consumption. Several experimental studies have found alcohol consumption to reduce the immediate effects of stress in a laboratory setting. We aimed to examine whether the association between alcohol and stroke depends on level of self-reported stress in a large prospective cohort. METHODS: The 5,373 men and 6,723 women participating in the second examination of the Copenhagen City Heart Study in 1981-1983 were asked at baseline about their self-reported level of stress and their weekly alcohol consumption. The participants were followed-up until 31st of December 1997 during which 880 first ever stroke events occurred. Data were analysed by means of Cox regression modelling. RESULTS: At a high stress level, weekly total consumption of 1-14 units of alcohol compared with no consumption seemed associated with a lower risk of stroke (adjusted RR: 0.57, 95% CI: 0.31-1.07). At lower stress levels, no clear associations were observed. Regarding subtypes, self-reported stress appeared only to modify the association between alcohol intake and ischaemic stroke events. Regarding specific types of alcoholic beverages, self-reported stress only modified the associations for intake of beer and wine. CONCLUSIONS: This study indicates that the apparent lower risk of stroke associated with moderate alcohol consumption is confined to a group of highly stressed persons. It is suggested that alcohol consumption may play a role in reducing the risk of stroke by modifying the physiological or psychological stress response.

Insular lesions, ECG abnormalities, and outcome in acute stroke.

It has been suggested that lesions in the insula may result in abnormal electrocardiographic (ECG) findings and increase the risk of sudden death. We investigated if computed tomography (CT) detected insular lesions due to acute stroke were related to ECG abnormalities and mortality at three months. Acute insular lesions were diagnosed in 43/179 patients (left insular = 25; right insular = 17; bilateral = 1) with acute stroke (cerebral infarcts = 62 and intracerebral haemorrhage = 17) based on CT scans from 5-8 days after stroke onset; 12 lead ECGs were recorded on admission and ECG telemetry was done in the first 12-24 hours after admission. Information regarding mortality at three months was obtained. Insular lesions were related to sinus tachycardia with heart rate >120 bpm (p = 0.001), ectopic beats >10% (p = 0.032), and ST elevation (p = 0.011). Right insular lesions were related to atrial fibrillation (p = 0.009), atrioventricular block (p = 0.029), ectopic beats >10% (p = 0.016), and inverted T wave (p = 0.040). Right insular lesions, compared with left or no insular lesions, increased the odds of death within three months (OR 6.2, 95% CI 1.5 to 25.2) independent of stroke severity, lesion volume, and age. As the number of patients in the present study is relatively small, our findings need to be confirmed in studies on other populations of stroke patients.

Glycerol for acute stroke.

BACKGROUND: Brain oedema is a major cause of early death after stroke. A 10% solution of glycerol is a hyperosmolar agent that is claimed to reduce brain oedema. OBJECTIVES: To determine whether intravenous (I.V.) glycerol treatment in acute stroke, either ischaemic or haemorrhagic, influences death rates and functional outcome in the short or long term, and whether the treatment is safe. SEARCH STRATEGY: The Cochrane Stroke Group trials register was searched (January 2003), and some trialists were personally contacted. SELECTION CRITERIA: All completed, randomised and quasi-randomised, controlled, published and unpublished comparisons, evaluating clinical outcome in which I.V. glycerol treatment was initiated within the first days after stroke onset. DATA COLLECTION AND ANALYSIS: Two reviewers independently applied the inclusion criteria, assessed the trial quality and extracted data and this was checked with all co-reviewers. Death from all causes, functional outcome, and adverse effects were analysed. MAIN RESULTS: Eleven completed, randomised trials comparing I.V. glycerol and control were considered. Analysis of death during the scheduled treatment period for acute ischaemic and/or haemorrhagic stroke was possible in 10 trials where 482 glycerol treated patients were compared with 463 control patients. Glycerol was associated with a non-significant reduction in the odds of death within the scheduled treatment period (Odds Ratio (OR) 0.78, 95% Confidence Intervals (CI) 0.58 to 1.06). Among patients with definite or probable ischaemic stroke, glycerol was associated with a significant reduction in the odds of death during the scheduled treatment period (OR 0.65, 95% CI 0.44 to 0.97). However, at the end of the scheduled follow up period, there was no significant difference in the odds of death (OR 0.98, 95% CI 0.73 to 1.31). Functional outcome was reported in only two studies but there were non-significantly more patients who had a good outcome at the end of scheduled follow up (OR 0.73, 95% CI 0.37 to 1.42). Haemolysis seems to be the only relevant adverse effect of glycerol treatment. REVIEWERS' CONCLUSIONS: This systematic review suggests a favourable effect of glycerol treatment on short term survival in patients with probable or definite ischaemic stroke but the confidence intervals were wide and the magnitude of the treatment effect may be only minimal. Due to the relatively small number of patients, and that the trials were performed in the pre-CT era, the results must be interpreted cautiously. The lack of evidence of benefit in long term survival does not support the routine or selective use of glycerol treatment in patients with acute stroke.

Teaching of neuroepidemiology in Europe: time for action.

Many epidemiological and clinical studies in Europe, especially in Eastern Europe and countries in transition, are of poor methodological quality because of lack of background knowledge in clinical epidemiology methods and study designs. The only way to improve the quality of epidemiological studies is to provide adequate undergraduate and/or postgraduate education for the health professionals and allied health professions. To facilitate this process, the European Federation of Neurological Societies (EFNS) Task Force on teaching of clinical epidemiology in Europe was set up in October 2000. Based on analyses of the current teaching and research activities in neuroepidemiology in Europe, this paper describes the Task Force recommendations aimed to improve these activities.