Prevalence of cardiovascular disease (CVD) in women shows regional variations not explained by common risk factors. Analysis of CVD incidence will provide insight into whether there is further divergence between regions with increasing age.Seven-year follow-up data on 2685 women aged 59-80 (mean 69) at baseline from 23 towns in the UK were available from the British Women's Heart and Health Study. Time to fatal or non-fatal CVD was analyzed using Cox regression with adjustment for risk factors, using multiple imputation for missing values.Compared to South England, CVD incidence is similar in North England ) and Scotland ), but lower in Midlands/Wales ). Event severity influenced regional variation, with South England showing lower fatal incident CVD than other regions, but higher non-fatal incident CVD. Kaplan-Meier plots suggested that regional divergence in CVD occurred before baseline (before mean baseline age of 69).In women, regional differences in CVD early in adult life do not further diverge in later life. This may be due to regional differences in early detection, survivorship of women entering the study, or event severity. Targeting health care resources for CVD by geographic variation may not be appropriate for older age-groups. Geographical variations in coronary heart disease (CHD) and stroke (together termed cardiovascular disease (CVD) here) have been identified and reported for a range of countries in terms of both prevalence -16 and iMethods for the BWHHS have been published previously , and werMulti-centre  and local research ethics committees provided approval for the study and informed consent was obtained from the women to complete the measurements used in this study, abstract information from medical records, link to the National Health Service Central Register and store data.th September 2007 for all sources. CVD deaths were indicated by ICD10 codes I200-259, I516, I600-679, I690-699, G460-469, and G450-3 (underlying or otherwise). Self-reported strokes were only included where symptoms were >24 hours  mis-reported as stroke). For potential events identified by review of medical records, a number of criteria were applied in order to count an event as incident CVD for analysis. In doubtful cases a telephone discussion was held with the woman's general practitioner to confirm or reject CVD diagnoses. For MI, at least one of the following was required: (1) ECG evidence of MI, (2) raised cardiac enzyme (including troponin), (3) hospital letter confirming MI. For stroke, at least one of the following was required: (1) report of ischaemic/haemorrhagic stroke on CT or MRI scan, (2) hospital letter confirming stroke, (3) final diagnosis from GP notes is ischaemic/haemorrhagic/subarachnoid/other stroke (in absence of scan and letter). For unstable angina a hospital letter confirming the diagnosis was required.Cardiovascular disease (CVD) was defined as any of angina, unstable angina, myocardial infarction or stroke. Prevalent events were informed by either self-report at baseline or medical record review events dated prior to baseline. Incident events were informed by either self-report at the three- or seven-year follow-up, medical record review, or death certificate, using a cut-off of 30Dates of CVD events were required for time-to-event analysis. Exact dates were available for events ascertained by record review or death certificates. In the case of events only ascertained by self-report , only a year was given; 31st December was therefore used for the purposes of analysis (those with missing year are excluded (n = 47 where no other incident event occurred)). For prevalent events, the date of the earliest pre-baseline event was used for analysis. Similarly for incident events, the date of the earliest post-baseline event was used.In addition to age, data on a variety of known and potential risk factors for CVD were collected at baseline. Smoking status , family history of CVD , alcohol intake , limited fruit intake (both winter and summer fruit intake less than once a week/never), limited physical activity (never take regular exercise), socioeconomic position, and CVD medications , 2) and leg length were obtained through the nurse examination. The presence of diabetes at baseline was identified from self-report (n = 31), record review (n = 19), both self-report and record review (n = 182), fasting glucose higher than 7 mmol/l only (n = 184), or diabetic medicines only (n = 1)). Blood samples were taken after a minimum eight hour fast and were used to obtain fasting glucose, total cholesterol, HDLc and triglycerides[Systolic blood pressure (SBP), waist circumference, obesity  of those alive at seven-year follow-up) or non-return of record review (313/3730 (8%) of those alive at final record review) is not considered here. However, loss of information regarding non-fatal events is expected to be low since loss to follow-up for both sources is small .Regional differences in both fatal and non-fatal CVD incidence were analysed using Cox regression adjusted for age. Individuals were censored on the date of emigration, date of death, or 30Further adjustment was made for known and potential risk factors . Ten datasets were imputed using all other factors (including age and region) to impute missing values. Log survival time and binary indicator for event were also included as predictors . Linear The characteristics of 4286 baseline responders are given in Table In total there were 549 deaths in baseline responders 12.8%) in the follow-up period. Of these, 150 were in South England (11.4% of baseline responders), 117 in Midlands/Wales (15.2%), 210 in North England (12.7%) and 72 in Scotland (13.2%). CVD prevalence at baseline and incidence over the seven year follow-up period for the 4826 responders at baseline is given in Table .8% in thResults of analysis for all incident events  are given in Table Analyses carried out separately for fatal and non-fatal incident CVD provides further insight into the nature of regional variations. The incidence of fatal CVD events only was, after adjusting for all considered covariates and missing data, higher in all the regions compared to South England: Midlands/Wales HR 1.13 (95% 0.72 to 1.77), North England HR 1.19 (95% CI 0.82 to 1.74), Scotland HR 1.09 (95% CI 0.66 to 1.82). In contrast, incidence of non-fatal events was lower in the Midland/Wales (HR 0.82 (95% CI 0.58 to 1.15) and Scotland (HR 0.86 (95% CI 0.58 to 1.28) compared to South England (adjusting for covariates and missing data).The Kaplan-Meier survival functions by region for CVD incidence are shown in Figure further divergence by region, although the differences by region remain at age 76 (corresponding to the mean age at seven-year follow-up). These results suggest that the regional differences in CVD occur at earlier ages; while examination of CVD prevalence is likely to show regional variation across a range of ages, regional differences in CVD incidence may only be apparent at younger ages. This possibility is supported by the original analysis of the regional differences in incidence data among men at much younger ages (40-59), which showed marked north-south variation [Evidence from the BWHHS published previously indicated that region was associated with CVD prevalence over and above variation explained by known risk factors, with South England having the lowest prevalence . Resultsariation .divergence of the regions in terms of CVD may arise for a number of reasons. There may be regional differences in terms of early diagnosis or treatment of conditions such as angina, which may arise due to differences in health care provision or access to/use of health care services. Such differences in diagnosis would influence the cohort at earlier ages, but may have relatively little impact later on. If this were the case, regions exhibiting higher CVD at younger ages may actually represent better clinical practice . However, since these results are adjusted for CVD medication, it seems unlikely that differences in early detection account for regional differences in CVD incidence. The potential impact of improved healthcare on survival following a CVD event implies that regions with higher non-fatal CVD incidence also show lower fatal CVD incidence (as observed here in South England).The absence of continued Survivorship may also play a role. Although prevalent non-fatal CVD events are included in analysis, fatal CVD events  occurring prior to baseline are not, since by definition an individual must be alive to participate in the study. Those still alive at the start of the study will thus be a healthier group. The impact of this survivorship may vary between regions since mortality (CVD and other causes) may differ between regions prior to baseline. This would imply that regions exhibiting lower CVD prevalence in fact represent a group from which more of the least healthy individuals have already died.It may also be possible that a different type of individual is at risk of early CVD . If this early onset CVD differed by region, but later onset CVD was more similar between regions, this would be consistent with the observations described here. Lastly, it is possible that there are regional differences in response to recent guidance on the management of cardiovascular risk factors and both primary and secondary prevention.Regional differences in cardiovascular disease incidence and mortality have been reported for a number of other countries, including the United Kingdom , the UniOne of the potential weaknesses of this analysis is missing data. This problem has two components, since there are missing data in the CVD risk factors included as covariates in the Cox models, and also non-response at each of the three follow-up time-points. The former is addressed through the use of multiple imputation, which assumes that the missing covariate values are missing at random (MAR) - that the missing values depend on observed values of other variables, but not on the missing values themselves. Given the well-documented strong relationships between many of the covariates of interest here, this would seem a generally reasonable assumption. Furthermore, imputation based on non-missing covariates is reasonable since individuals do not generally have many missing covariates  have at least 9/15 non-missing values). The results from analysis of the multiply imputed datasets suggest that accounting for the missing data explains some of the observed differences between regions.2 p = 0.09) or seven-year follow-up (χ2 p = 0.18). Non-response at baseline however is more pronounced than at subsequent waves  and there is strong evidence for a difference in response at baseline by region (χ2 p < 0.0005). Since non-responders at baseline are excluded from all analyses, this has implications for bias and generalisability of the results presented here. If regions with higher non-response at baseline actually have higher CVD incidence, the large number of non-responders (those at likely to be at highest risk of CVD) would render estimates of regional differences conservative.The second component of the missing data issue is non-response to either the baseline or follow-up questionnaires. Non-response to the follow-up questionnaires is not addressed here, since it is anticipated that the majority of self-report CVD events missed as a result of this non-response would be picked up by record review . Furthermore, there is little evidence of a difference in response by region at either three-year  occur relatively early in life, but do not continue to diverge in later life. Analysis of the incidence of fatal and non-fatal CVD combined showed different patterns of variation between the regions, suggesting regional differences in prognosis following a CVD event. These findings imply that in women, efforts to characterise and standardise regional differences in CVD-related healthcare should be targeted at those under 70. Further follow-up of the BWHHS is required in order to increase numbers of events and thus increase the precision of the estimates obtained in these analyses. Further work is also needed to investigate potential explanations for the apparent improvement in CVD outcomes observed in South England compared to other regions.The authors declare that they have no competing interests.LK carried out the analysis and wrote and revised the manuscript. SE, DL and CC helped draft and revise the manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/696/prepubBaseline questionnaire. A self administered questionnaire about lifestyle and medical history used at the baseline visit.Click here for file3-year follow-up questionnaire (long form). A self administered questionnaire about lifestyle and medical history which was sent out in March 2003.Click here for file3-year follow-up questionnaire (short form). A self administered questionnaire about lifestyle and medical history which was sent out in March 2003.Click here for file7-year follow-up questionnaire. A third self-administered postal questionnaire was sent in 2007. This was about health and lifestyle to see what was changed, and what has stayed the same since the last survey in 2003.Click here for file


Evidence-based guidelines for the management of hypertension are now well established. Studies have shown that more than 60% of patients with hypertension will require two or more drugs to achieve current treatment targets.Combination therapy is recommended as first-line treatment by the JNC-7 guidelines for patients with a blood pressure > 20 mmHg above the systolic goal or 10 mmHg above the diastolic goal, while the International Society of Hypertension in Blacks recommends combination therapy when BP exceeds targets by > 15/10 mmHg. Current European Society of Hypertension-European Society of Cardiology guidelines also recommend the use of low-dose combination therapy in the first-line setting. Furthermore, JNC-7 recommends that a thiazide-type diuretic should be part of initial first-line combination therapy. Thiazide/diuretic combinations are available for a variety of classes of antihypertensive, including ACE inhibitors, angiotensin receptor blockers (ARBs), beta blockers and centrally acting agents. This article focuses on clinical data investigating the combination of an ARB, irbesartan, with the diuretic, hydrochlorothiazide.These data indicate that the ARB/HCTZ combination has greater potency and a similar side effect profile to ARB monotherapy and represents a highly effective approach for attaining goal BP levels using a therapeutic strategy that very effectively lowers BP, is well tolerated and minimises diuretic-induced metabolic effects.The PubMed and other searchable databases were utilized to collate information from original and review articles as well as from selected abstracts relevant to this topic.Diuretic-based combination antihypertensive drug therapy is a cornerstone of antihypertensive drug therapy. Most hypertensive patients will require more than one antihypertensive drug to lower blood pressure (BP) below target levels. The combination of diuretics with renin angiotensin system antagonists is highly logical given the significant augmentation of BP response and the minimization of drug-specific side effects  when these two drug classes are combined. The combined use of angiotensin receptor blockers and diuretics is better tolerated, but more costly, than generic angiotensin converting enzyme inhibitors and diuretics, mostly because of the absence of cough and much lower incidence of angioedema. Hypertension affects almost 29% of the adult US population, an estimated 58.4 million individuals . WorldwiMaintaining aggressive BP targets is the basis of preventing the long-term adverse outcomes of hypertension. The linkage of efficacious and prompt treatment has been suggested by the results of the VALUE trial, which was designed to compare the incidence of cardiac morbidity and mortality when the same level of BP control was achieved using treatment regimens based on the angiotensin receptor blocker (ARB) valsartan or the calcium channel blocker amlodipine . HoweverWhile BP goal attainment is the most crucial objective of antihypertensive treatment, other considerations are also of fundamental importance. One key consideration is that BP control should be sustained over 24 h. Long-acting antihypertensive agents provide 24-h BP control from a single daily dose, as well as attenuating the early morning rise in BP . Long-acMore than two-thirds of hypertensive individuals will require two or more antihypertensive agents selected from different drug classes to achieve their BP targets ,14. For JNC-7 recommends that combination therapy should usually include a thiazide-type diuretic as first-line therapy for stage 2 hypertension as well as for patients with compelling indications ,19. EquaAngiotensin receptor blockers have an established record of BP-lowering efficacy and a placebo-like side effect profile in hypertensive patients. Recent outcome studies in high-risk hypertensives have shown that ARBs provide cardiovascular-renal protection beyond what can be entirely attributed to BP-lowering alone. In hypertensives with ECG-LVH, there was a lower incidence of stroke in the losartan (ARB) than the atenolol (beta-blocker) treatment arm despite attainment of virtually identical BP lowering as determined by cuff measurements . LosartaNotwithstanding these benefits, ARBs like other classes of antihypertensive do not provide sufficient BP control for many patients when used in monotherapy. Indeed, ARBs, similar to ACE inhibitors, have near-flat dose–response curves suggesting that monotherapy dose titration offers limited benefits. This was shown in a pooled meta analysis of 43 published randomised clinical trials of losartan, valsartan, irbesartan and candesartan when administered at doses recommended for the treatment of hypertension . ARB monIn addition to their impressive combined BP-lowering effects, the use of ARBs and HCTZ in combination counteracts the potential adverse effects of these agents when given as monotherapy. Because of the tendency of ARBs to elevate potassium levels, this is less likely to be a problem when combined with diuretics . LikewisTolerability considerations also support the use of ARBs rather than ACE inhibitors in combination therapy. For instance, in the recent Blood Pressure Reduction and Tolerability of Valsartan in Comparison With Lisinopril (PREVAIL) trial, patients with mild-to-moderate hypertension treated with ARB/HTCZ combination were less likely to experience adverse events than those treated with ACE-inhibitor/HTCZ combination therapy . Drug-related dry cough also occurred more frequently in the patients receiving ACE inhibitor (7.2%) than in those receiving ARB (1.0%) . AngioedFixed-dose combinations of HCTZ and ARBs provide effective, simple, aggressive and well-tolerated BP control and are now rapidly gaining acceptance with physicians. The usefulness of ARB/HCTZ combinations in hypertension has now been demonstrated in clinical trials for most of the ARBs –47. SomeIn the following section, the data for another representative ARB, irbesartan, are reviewed to explore in greater detail the clinical potential of the ARB/HCTZ strategy.The safety and BP-lowering effect of the combination of irbesartan plus HCTZ, administered orally once a day, in patients with hypertension have been investigated in several clinical trials . The irbThe long-term safety and antihypertensive effects of irbesartan/HCTZ have also been demonstrated . HyperteAnother study used a 4 × 4 factorial design or ‘matrix’ design to evaluate irbesartan and HCTZ across their respective dose ranges in patients with mild-to-moderate hypertension . A totalThe combination of an ARB and HCTZ has been reported to be effective in difficult-to-treat and severely hypertensive patients for several ARBs, including losartan, candesartan, telmisartan and eprosartan –58. A hiThe IrbesartaN/HCTZ bLood pressUre reductionS in dIVErse populations (INCLUSIVE) trial was a multicenter, prospective, open-label, single arm study to evaluate the BP-lowering efficacy and safety of a fixed-dose combination of irbesartan and HCTZ, at low dose (150 mg/12.5 mg) and at high dose (300 mg/25 mg) sequentially for 8 weeks each. Each combination of irbesartan and HCTZ was administered as a single tablet. Treatment was given to over 800 difficult-to-treat hypertensive patients including the elderly people, African-Americans, Hispanics, patients with type 2 diabetes and patients with the metabolic syndrome . OverallIrbesartan has also been investigated in severely hypertensive patients. In a randomised, double-blind study of 737 patients with severe hypertension, 468 patients were given fixed-dose irbesartan/HCTZ 150/12.5 mg for 1 week then were force-titrated to a 300/25 mg dose for a further 6 weeks . The remAngiotensin receptor blockers used in monotherapy have been widely reported to vary in their BP-lowering efficacy. For instance, irbesartan at a starting dose of 150 mg has been reported to reduce BP more effectively than valsartan at its original starting dose of 80 mg . HoweverSimilar differences in BP-lowering efficacy have been reported in the fixed-dose combination setting, although the same difficulties with dose selection exist. The recent COmparative Study of efficacy of Irbesartan/HCTZ with valsartan/HCTZ using home blood pressure Monitoring in the treAtment of mild-to-moderate hypertension (COSIMA) study demonstrated greater antihypertensive efficacy of the fixed-dose combination of irbesartan 150 mg/HCTZ 12.5 mg vs. a fixed-dose combination of valsartan 80 mg/HCTZ 12.5 mg in an 8-week study of 414 hypertensive patients uncontrolled on HCTZ , althougDifferences in antihypertensive efficacy have also been reported between various other ARB/HCTZ combinations. For instance, in a recent 8-week study in 1066 patients, telmisartan/HCTZ 80/25 mg was reported to reduce both systolic and diastolic BP to a greater degree than valsartan/HCTZ 160/25 mg . Once agIn conclusion, it remains difficult to rank ARB/HCTZ combinations in terms of basic BP-reducing efficacy, as relatively few head-to-head trials have been carried out and the majority have not compared agents at like-for-like doses and with respect to universally agreed end-points. The goal of treatment should therefore be to treat patients to target irrespective of which particular ARB/HCTZ combination is used, with prompt dose escalation and the addition of a third agent whenever necessary to achieve this goal.This study highlights an intriguing aspect of therapy with angiotensin receptor blockade. That is, the dose–response curves for BP-lowering and target-organ protection are not identical. In this double-masked, randomised, crossover study in 52 hypertensive persons with type 2 diabetes and microalbuminuria, all treated with bendroflumethiazide 5 mg/day, irbesartan was also administered in doses of 300, 600, and 900 mg/day . UrinaryThere is a need for effective, safe and simple therapies to treat hypertension to recommended BP targets rapidly and rigorously, but with good tolerability and sustained patient adherence. The use of combination therapy as first-line treatment will help more patients promptly achieve BP goals, and fixed-dose combinations provide a means for simple but flexible dosing. Combination therapy using ARBs with HCTZ provides greater potency and fewer side effects than higher-dose monotherapy with either agent, and potentially offers benefits beyond those of BP lowering alone, particularly in high-risk hypertensives. The results from the irbesartan/HCTZ studies illustrate the potential of ARB/HCTZ therapy as a starting treatment in patients with moderate and severe hypertension; in patients with stage 2 disease such treatment enables management goals specified by current guidelines to be more rapidly and effectively achieved.


Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions. Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses . CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions. Human basophils, as other leukocytes, express several cell membrane antigens which can be related to their immunological responsiveness. Challenging basophils with allergens or agonists may result in a modified expression of these molecules on cell membrane, a mechanism that can be evaluated by flow cytometry -5. FurthWhile most authors recognize the flow cytometric approach as a proper tool to investigate basophil function, several problems and methodological issues are still to be clarified, namely subject responsiveness (a broad variability in basophil activity is evidenced between different donors and different markers within the same donor), sample treatment (which may affect cell activation state and response pattern), gating procedure  and the selection of the best suitable activation markers -13. The expressing lymphocyte area: the use of CD45 contributes to discriminate basophil area from other leukocytes excluding cellular debris . Th. Thbrighbright cells increased from 3.12% to 33.44% following fMLP activation and from 3.12% to about 13.67% following anti-IgE stimulation. The N-aminopeptidase CD13 and the c-type lectin CD69 did not show, however, a reproducible activation pattern in our assay condition, which led us to focus on CD63 (lysosome-associated membrane protein-3 or LAMP-3) and CD203c (ecto-nucleotide-pyrophoshatase phosphodiesterase-3 or ENPP-3) markers hereafter.Table 1 [see Additional file Basophil response towards the different agonists was examined following changes in the mean fluorescence intensity (MFI) associated to specific membrane marker fluorochrome and by evaluating dot plots of the acquired events.Figures dim/CD203cbright phenotype is clearly evident. This might indicate: a) the presence of a CD203cbright basophil sub-population lacking CD63 tetraspanin or b) that the CD63 dimly cells have this intracellular-associated protein 3 but are unable to up-regulate it on the membrane. To examine these issues, we investigated intracellular storage of CD63. Figure non expressing phenotype on their membrane  while CD63 reached the maximum of activation at 3 × 10-8 M (changing MFI from 179 to 4020). Moreover, while CD63 up-regulation seems to go forward up to 10-6 M fMLP, CD203c reached a maximum plateau at the dose in which CD63 began to increase  samples of Ksiveness . This manon expressing/CD123bright gating strategy. Although CD203c is a selective marker for basophilic cells, we did not choose this molecule as phenotyping tracer since the brighter CD123 revealed as a better marker to separate cells and because we used the CD203c for activation studies.Basophil electronic capture was very efficient by using a HLADRBasophils appeared as highly responsive to agonist triggering in a time-course and dose-response manner. We explored several classic markers that were previously described in the literature ,14,34-36After basophil treatment with fMLP and with anti-IgE, an increase in MFI of CD63, CD203c, CD45 and CD13 was observed (the latter only with fMLP), while CD69, used by others as activation marker ,37-39, dWith anti-IgE and calcium ionophore as agonists, the two markers behaved in a similar way in the dose-response curve, suggesting that the discrepancy between the two markers, noted with fMLP, is receptor-specific and does not belong to a calcium-activated pathway. It is tempting to speculate that fMLP, being both a chemotactic factor and a degranulating agonist, requires some specific regulation, which may be more sophisticated and flexible than those required by anti-IgE, the latter being a substance that mimics the physiologic way of basophil activation that induces the histamine release. Interestingly, in human neutrophils intracellular calcium release and actin polymerization require fMLP doses much lower than those necessary for superoxide production activation and lysozyme release ,46, suggIn summary the results from this multiparametric approach suggest:a) CD63 is the most sensitive and early activation marker of human basophils, in agreement with previous evidence from literature. However, CD63 is expressed in only a minority of basophils even at optimal agonist doses, while it is present in all the cells as intracellular pool.b) CD203c is less sensitive as activation marker, because there is a substantial expression also in the resting state. On the other hand, this marker is upregulated in all the cells following activation, also in those that are not identified as activated using CD63.c) The dose-response curves show a receptor-specific  dissociation between the two activation markers. This evidence suggests that the molecule marked by CD203c is associated with the low-dose events of chemotaxis while the molecule CD63 is associated with degranulation, as previously reported.d) An overall view of several activation molecules within the same experiment, using pooled cell preparations could be an affordable tool to investigate the pharmacology of basophil activation. Testing other basophil agonists and deepening the knowledge of the transduction pathways and of gene expression may substantiate these views.The authors declare that they have no competing interests.SC and PB conceived the study, performed the experiments and wrote the manuscript, AV and RO assisted in the flow cytometry setting and handling, MDG and PS participated in the subjects recruitment and in blood specimen selection and GT participated in study design and coordination. All authors read and approved the final manuscript.Mean of fluorescence intensity (MFI) of basophil markers in resting and agonist stimulated human basophils. The data provided represent the statistical analysis of various membrane markers of human basophils.Click here for file


Anti-TNF therapies have revolutionized the treatment of rheumatoid arthritis (RA), a common systemic autoimmune disease involving destruction of the synovial joints. However, in the practice of rheumatology approximately one-third of patients demonstrate no clinical improvement in response to treatment with anti-TNF therapies, while another third demonstrate a partial response, and one-third an excellent and sustained response. Since no clinical or laboratory tests are available to predict response to anti-TNF therapies, great need exists for predictive biomarkers.Here we present a multi-step proteomics approach using arthritis antigen arrays, a multiplex cytokine assay, and conventional ELISA, with the objective to identify a biomarker signature in three ethnically diverse cohorts of RA patients treated with the anti-TNF therapy etanercept.We identified a 24-biomarker signature that enabled prediction of a positive clinical response to etanercept in all three cohorts .We identified a multi-parameter protein biomarker that enables pretreatment classification and prediction of etanercept responders, and tested this biomarker using three independent cohorts of RA patients. Although further validation in prospective and larger cohorts is needed, our observations demonstrate that multiplex characterization of autoantibodies and cytokines provides clinical utility for predicting response to the anti-TNF therapy etanercept in RA patients. Rheumatoid arthritis (RA) is a prototypical systemic autoimmune disease that affects 1% of the world population. TNF antagonists have become the most widely used biological therapies for patients with RA , 30 to 5Great need exists for molecular biomarkers for the prediction of response to anti-TNF therapies, and a number of candidate markers are currently under investigation, including genetic and protein markers . RA is aWe previously reported the development of antigen microarrays and application of these arrays to characterize autoantibody phenotypes associated with a variety of autoimmune diseases . We furtIn the present report, we describe application of a multi-step proteomics approach using RA antigen arrays and cytokine arrays to discover and validate a multivariable biomarker for prediction of response to the anti-TNF therapy etanercept, using sera derived from three independent cohorts of patients with RA. The workflow of the studies is outlined in Figure ®, Amgen, Thousand Oaks, CA, USA), were analyzed using synovial antigen microarrays (except for the third cohort), ELISAs, and a multiplex 12-cytokine bead assay. The cytokines assayed were selected based on previous screening studies using a 22-cytokine assay [Pretreatment sera from three cohorts of patients with the diagnosis of RA based on the ACR classification criteria , who werThe three cohorts included 29 Caucasian patients from the ABCoN cohort of the North American Rheumatoid Arthritis Consortium (US cohort), 43 Caucasian patients seen at Swedish tertiary care centers and collected through the Karolinska-lead EiRA initiative (Swedish cohort), and 21 Japanese patients (Japanese cohort). The patients' demographic, clinical and serologic characteristics are summarized in Table Blood samples were obtained at baseline (pretreatment sample) and at least 3 months after initiation of therapy with etanercept. Analysis of the pretreatment samples was performed for the present study. Response to therapy with etanercept was assessed at least 3 months after etanercept was started, based on the ACR criteria for improvement . All sam® was added to achieve a final concentration of 3 μg/ml, as previously described in detail [® kit  that included beads specific for TNFα, IL-1α, IL-1β, IL-6, IL12p40, IL-12p70, IL-15, granulocyte- macrophage colony-stimulating factor, fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), eotaxin, and IFNγ-inducible protein 10.All cytokine measurements were performed using the Luminex ×200 platform, following a previously described optimized assay protocol . Brieflyn detail . For the® microscope slides  using a robotic arrayer. The arrays were blocked and probed with sera at 1:200 dilutions, and bound serum autoantibodies detected using Cy3-conjugated goat anti-human IgG secondary antibodies . Probed arrays were scanned with a GenePix 4000B scanner , and antibody reactivities quantified using GenePix Pro 5.0 software.The production of RA antigen microarrays was previously described in detail ,14. BriePeptides cfc(48–65)cit1 .Peptides and fibrinogen protein from human plasma  were coated onto medium-binding 96-well flat-bottom polystyrene plates  at 1 μg peptide/ml at 4°C overnight. Plates were then washed and blocked for 1 hour at room temperature using 5% dry milk in PBS, and incubated for 90 minutes using serum at 1:200 dilution in PBS. Horseradish peroxidase-conjugated anti-human IgG secondary antibodies  were used at 1:20,000 dilutions, and bound autoantibodies were detected by chemoluminescence  (version 1.21) and prediction analysis of microarrays (PAM) (version 1.23), and the hierarchical clustering software ClusterreeView® , as descreeView® ,17. PAM reeView® . PAM wasreeView® .z-normalization procedures were applied.Non-normalized datasets were used for all analyses in the article. Although this approach limits the ability to detect differences in the low signal intensity range, the rationale for this approach is based on the observation that high-level reactivities became significantly distorted when ®; Sigma Genosys, St Louis, MO, USA).To further develop previously-described RA autoantigen microarrays , we expaIn line with and expanding earlier results using smaller 225-antigen arrays, we observed on the > 540-antigen arrays specific and differential serum autoantibody reactivities to COMP, clusterin, osteoglycin, apolipoprotein E, histones H2A and H2B, serine protease 11, and other candidate antigens. Representative array images of antibody reactivities are shown for sera from two different patients with RA; one patient that exhibited low serum autoantibody reactivity Figure  and anotR2 ranging from 0.49 for clusterin(386–405)cit to > 0.92 for hFibA(211–230)cit  ≤ 4.3%) is presented in Figure ® software). A compiled list of the top-scoring antigens identified in multiple screening experiments is presented in Table We screened 29 pretreatment serum samples from the ABCoN cohort using RA antigen microarrays. SAM identified differential pretreatment levels of autoantibodies that were present at elevated levels in etanercept responders (response ≥ ACR50) as compared with nonresponders . A representative result of the top-scoring SAM-identified autoantigens . Based on these preliminary observations, combined autoantibody and cytokine analyses were used in the subsequent experiments outlined below with the objective to develop a multi-parameter biomarker for predicting response to etanercept therapy.To determine whether combinations of cytokines and autoantibodies might provide superior differentiation of pretreatment samples derived from etanercept responders and nonresponders, we next performed SAM analysis on integrated antigen array and cytokine datasets obtained for the 29 ABCoN samples. This analysis demonstrated that a panel of antigens and cytokines more effectively differentiated baseline samples derived from responders and nonresponders , while MCP-1 and IL-6 exhibited a trend towards being elevated in responders as compared with nonresponders (P < 0.1).In a first step, concentrations of 12 cytokines were measured and analyzed by logistic regression in all 93 pretreatment samples derived from the three independent cohorts. When cytokine results from baseline samples derived from responders and nonresponders were compared, TNF and IL-15 were elevated in responders as compared with nonresponders  values were used for further analyses. Since relative expression levels were the primary measure of interest for these assays, no standard curves for calculation of antibody concentrations were developed. All measurements from the single-antigen ELISAs were combined with the measurements from the bead-array cytokine assay for integrated analysis of cytokine and autoantibody profiles in baseline samples derived from all three cohorts of etanercept new-start RA patients.T(0.14)) , and the PAM rank list of these parameters including their corresponding scores for responders and nonresponders are shown in Figure PAM with error plots of training and cross-validation are shown in Figure T(0.14), and to determine its utility for prediction of response in each cohort independently, the 24-parameter classifier was then applied to the three cohorts individually. Prediction probabilities were calculated for the responder and nonresponder classes. Classification errors as well as positive predictive values and negative predictive values are shown in the corresponding tables for each cohort , and to predict lack of response to etanercept therapy (response < ACR20). This biomarker signature enabled superior pretreatment classification of response in three ethnically diverse cohorts, in comparison with a theoretical benchmark based on clinical observation and previous experience in population-based cohorts .Identification of clinical predictors and development of molecular biomarkers have been hampered by many factors, including the molecular complexity and clinical heterogeneity of RA, the inherent difficulty in classifying response to therapy that appears random and does not follow a Gaussian distribution , and theTo address this unmet clinical need and some of the above-mentioned limitations, we applied proteomics technologies to characterize pretreatment samples from three independent cohorts, whereby all patients were treated with a single anti-TNF therapy (etanercept).Using data from all three cohorts, we identified a panel of proteins, characterized by elevations of both serum antibody and cytokine concentrations, which were associated with patients who exhibited clinically good response (≥ ACR50) and excellent response (≥ ACR70) to etanercept therapy . In contrast, patients who exhibited minimal or no significant response to etanercept therapy after 3 months or more were found to predominantly lack this biomarker signature .The RADIUS program – the Rheumatoid Arthritis DMARD Intervention and Utilization Study , which fOur study investigated samples from three population-based cohorts. Within the US-based ABCoN cohort, for which the multi-parameter biomarker demonstrated the best response prediction, analysis of the responders exhibiting an ACR50 response or greater provided a positive predictive value of 71% Figure . In the Some of the markers in the 24-variable biomarker signature were previously proposed to have some value for prediction, such as MCP-1  and titevalidation in independent cohorts of the identified markers is not presented in this paper. Because of the small sample numbers in the individual cohorts and the fact that the experiments were performed over a 3-year period, the present article represents a discovery and testing study. The training was performed on the full dataset, and the resulting panel of markers was tested in the three individual cohorts. These limitations indicate the need for larger validation studies and prospective serum proteome studies in cohorts where longer-term response outcomes are available.Although the present study utilizes samples from three cohorts of patients, important limitations remain: the number of samples per cohorts is relatively small; a number of samples demonstrated undetectable serum levels of cytokines; the quantification of response to therapy in RA is intrinsically inaccurate since response classification is based on short term follow-up; and our data may underestimate the predictive value of these biomarkers for predicting response to anti-TNF therapy since concomitant disease-modifying anti-rheumatic drug medication in the three cohorts was not uniform. Moreover, a full We identified a 24-parameter autoantibody and cytokine biomarker that enables pretreatment classification and prediction of etanercept responders in three cohorts of patients with RA. Although further validation in prospective and larger cohorts is needed, it is proposed that the multi-parameter protein biomarker presented in this study will facilitate the development of molecular biomarkers with even higher predictive utilities for guiding use of anti-TNF therapy and other disease-modifying therapies in RA patients.ACR: American College of Rheumatology; COMP: cartilage oligomeric matrix protein; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; MCP-1: monocyte chemoattractant protein-1; PAM: prediction analysis of microarrays; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; SAM: significance analysis of microarrays; TNF: tumor necrosis factor.The authors declare that US and international patent applications were filed for biomarkers to guide selection of therapy in RA, and option agreements for this intellectual property have been signed with third-party companies.WH and WHR conceived of the study and designed the arthritis antigen arrays. PAM performed immunoprecipitation experiments and mass spectrometry studies. WH and BHT carried out the microarray experiments, and multiplex cytokine and ELISA experiments. RJT, WL and BHT performed the statistical analysis. FB, RFvV, JL, LK, YT, KS, PKG and MCG participated in design and coordination. WH drafted the manuscript. All authors read and approved the final manuscript.Excel file containing a table that summarizes the clinical characteristics of the Japanese cohort, including co-medications.Click here for filex axis and optical density values are plotted on the y axis. Coefficients of linear regression analysis (R2) are shown for each peptide.Adobe Illustrator file containing a figure that shows the analysis of microarray reactivities by conventional ELISA. Array-determined digital fluorescence units (arrayFU) are plotted on the Click here for fileAdobe Illustrator file containing a figure that demonstrates logistic regression analysis of the six other cytokines not shown in Figure Click here for file


Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR  (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format.During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15.We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping. Mycobacterium tuberculosis (MTB). IS6110-restriction fragment length polymorphism (RFLP) is the reference technique for genotyping MTB ) in the MIRU analysis. This decision was supported by studies that found epidemiological links for clusters including isolates with subtle differences in RFLP isolates [In this study, we considered full or high values as indicative of a good correlation between MIRU-15 and RFLP. Correlation was considered to be high when MIRU-grouped isolates sharing RFLP patterns showed high isolates -25 and blarity ≥ 5%, or isisolates ,19, whicIS6110 band similarity percentages with the other clustered isolates. The spoligotypes for these two isolates were also different from the R7 representative pattern, although they belonged to the same genetic lineage (T5 family). This could be due to the fact that MIRU-15 misassigned these isolates and we were probably observing a genotypic convergence phenomenon. On the other hand, the rate of MIRU changes is relatively lower than that of IS6110-RFLP and spoligotyping, so it could also be possible that the MIRU type in this case is shared by different representatives of the T5 lineage. In the remaining two noncorrelated clusters, we observed that MIRU-15 split them to a different extent, and we detected differences (in more than two loci) among isolates sharing an RFLP type. In addition, the largest RFLP cluster was fully split by MIRU-15  with differences in 2–5 loci for all the isolates. Splitting of RFLP clusters by MIRU-15 has been found in clusters involving strains with low-copy-band fingerprints (< 6 bands) [Although good correlation was generally found in our convenience sample, no correlation with RFLP was observed for three RFLP clusters. MIRU-15 added two isolates in the R7 RFLP cluster, which showed only low 6 bands) ,26,27, w6 bands) , and theIn order to fully understand the meaning of the concordances and discordances between RFLP and MIRU-15 data, we used an unselected population-based sample with available epidemiological data to interpret potential discrepancies. We chose the MTB isolates cultured in the province of Almería during a 2.5-year period to ensure quality in the clustering assignation and to increase the observation time of other studies . We deciAs with our findings in the convenience sample, the correlation between MIRU-15 and RFLP genotypes was good; full or high correlation with the RFLP data was detected for 24 of the 29 clusters genotyped by MIRU-15. We found epidemiological links in 72.7% of the clusters and in 84.3% of the cases with available epidemiological data. These percentages of "epidemiologically proved" clusters agree with those of other studies, and it is well known that higher values are only obtained if highly refined epidemiological surveys are followed up -30. It iThe data obtained by MIRU-15 in this study and elsewhere  suggest Clinical specimens were processed according to standard methods and grown in Lowenstein-Jensen slants and in MGIT  liquid media.IS6110-RFLP [This was composed of 134 MTB isolates from independent patients in three institutions in Almería (southeast Spain). The isolates had been previously analyzed using a set of 12 MIRU-VNTR loci and 110-RFLP .IS6110 copies.From January 2003 to June 2006, 353 MTB isolates were cultured from independent patients  from the three public hospitals in the province of Almería  and 308 of these (87.2%) were genotyped by RFLP. From this sample, we selected the 91 isolates clustered by RFLP. All the isolates analyzed had patterns with more than six IS6110-RFLP typing, and spoligotyping were performed according to standard methods [DNA extraction,  methods  with somThe MIRU-type was defined after combining the results for the 15 loci in the following order: 580 (MIRU4), 2996 (MIRU26), 802 (MIRU40), 960 (MIRU10), 1644 (MIRU16), 3192 (MIRU31), 424 (Mtub04), 577 (ETRC), 2165 (ETRA), 2401 (Mtub30), 3690 (Mtub39), 4156 (QUB4156), 2163b (QUB11b), 1995 (Mtub21), and 4052 (QUB26).IS6110 band similarity was observed between patterns. IS6110-RFLP, spoligotyping, and MIRU-VNTR dendrograms were generated using the unweighted pair group method with arithmetic averages (UPGMA). The Dice coefficient was used with RFLP and the categorical coefficient with spoligotyping and MIRU.Genotypes were analyzed using Bionumerics 4.6 . Clusters were defined when 100% IS6110-RFLP analysis was defined as follows:Correlation between MIRU-VNTR and IS6110 RFLP and MIRU shared identical patterns.a) Full correlation, when the isolates clustered by IS6110 band similarity (≥ 95%).b) High correlation, when we detected an SLV by MIRU  in the cluster defined by RFLP or some MIRU-clustered isolates not sharing identical RFLP genotypes but with a high c) No correlation, when RFLP-clustered isolates were clearly split by MIRU (differences in more than one locus) or MIRU-clustered isolates were clearly different by RFLP (similarities < 95%).The Hunter-Gaston discriminatory index (HGDI) was calculated as described elsewhere .In order to evaluate the epidemiological support of clusters in the population sample in Almería, we retrospectively analyzed data from the clinical charts and from standardized interviews with the patients. From these data we defined a scale of epidemiological evidence for the clustered cases based on Supply et al  as folloValue 1: proved, when the standard epidemiological survey or the interview of the clustered cases revealed the existence of proven links between the cases.Value 2: probable, when the standard epidemiological survey of the clustered cases revealed the existence of likely links between the cases.Value 0: improbable, when the standard epidemiological survey of the clustered cases revealed lack of links between the cases.Value -1: epidemiological data unavailable.NAR designed the study, performed all the MIRU-VNTR experimental assays, and was involved in the analysis of RFLP, spoligotyping, and MIRU-VNTR results. She analyzed the results and produced the first version of the manuscript. MML analyzed all the clinical and epidemiological data of the Almería sample and he was involved in the analysis of the correlations between RFLP and MIRU in the population sample. MH was involved in the analysis of the isolates by RFLP and spoligotyping. MSB and PB compiled and analyzed all the clinical and epidemiological data. EB critically reviewed the final version of the manuscript. DGV designed the study, supervised all the experimental work, analyzed the results, corrected and produced the final version of the manuscript.All authors read and approved the final manuscript.


A radioimmunoassay (RIA) for cytosine arabinoside (AraC) has been developed using antiserum raised in a sheep to an AraC monophosphate-ovalbumin conjugate. The antibody shows only 0.008% cross-reactivity with uracil arabinoside (AraU) and low (0.023%) cross-reactivity with other commonly co-administered drugs such as cytotoxic and antibacterial agents, and also a number of naturally occurring nucleosides and nucleotides. It does however cross-react by 125% with AraC monophosphate and by 109% with AraC triphosphate. As little as 1 ng/ml of AraC can be detected in plasma, serum, urine and cerebrospinal fluid (CSF) with no need for prior extraction. This RIA has been used to follow the disappearance of AraC from the plasma of patients receiving the drug.


Conventional antipsychotics augmented with benzodiazepines have been the standard acute treatment for psychiatric emergencies for more than 50 years. The inability of patients to give informed consent limits randomised, controlled studies. This observational study on immediate therapy for aggression and impulse control in acutely agitated patients (IMPULSE) evaluated the short-term effectiveness and tolerability of atypical and typical antipsychotic medications (AP) in a non-interventional setting.This was a comparative, non-randomised, prospective, open-label, observational study. Treatment over the first 5 days was classified according to whether any olanzapine, risperidone, or haloperidol was included or not. Documentations  were at baseline (day 1) and days 2–6 after start of AP.During the short treatment-period, PANSS-EC and CGI-aggression scores improved in all cohorts. 68.7% of patients treated with olanzapine, 72.2% of patients treated with risperidone, and 83.3% of patients treated with haloperidol received concomitant benzodiazepines . More patients treated with olanzapine (73.8%) were fully alert according to a tranquilisation score and active at day 2 than patients treated with risperidone (57.1%) or haloperidol (58.0%).Current medication practices for immediate aggression control are effective with positive results present within a few days. In this study, concomitant benzodiazepine use was significantly more frequent in patients receiving haloperidol. Behavioural emergencies occur in approximately 1% to 20% of psychiatric patients admitted to hospitals and necessitate physical restraint, seclusion and involuntary  medication . SeriousFor more than 50 years, conventional antipsychotics augmented with benzodiazepines have been the standard treatment ,5. The mThe IMPULSE study was a non-randomised, non-interventional, open-label, prospective, multi-centre observational study in an unselected sample of patients with behavioural emergencies. The study description asked for patients with schizophrenia, meeting the following criteria for participation: adult patients (≥ 18 years) newly admitted to a psychiatric or forensic hospital, presenting with agitation with or without aggression and requiring antipsychotic treatment. Patients were recruited in Germany between July 2003 and October 2004. Because this was a non-interventional, observational study according to German Drug Law (AMG) §67 (6), explicit ethical approval by an institutional review board or written patient consent was not required. The German Federal Institute for Drugs and Medical Devices  and the German Statutory Health Insurance of Physicians  were notified about this observational study prior to the start of data collection. Decisions on patient recruitment, the antipsychotic treatment given and use or non-use of benzodiazepines were completely at the discretion of the investigator. Due to the lack of randomisation and pre-defined recruitment rules, patients receiving olanzapine were over-represented when compared to the olanzapine market share  of antipsychotic agents in Germany during the recruitment period in 2003–2004. Moreover, the investigators also recruited patients with psychiatric disorders other than schizophrenia.Assessments were carried out daily, at baseline (day 1) and the following 5 days after initial documentation (days 2–6). Diagnoses were documented according to the International Classification of Diseases, 10th revision (ICD-10) . The prePsychiatric emergencies were categorised as follows: (i) Self-endangering behaviour included acute suicidal thoughts or plans, compulsatory admission because of suicidality as well as self-injuring behaviour; (ii) Third party endangering was defined to be present if patients presented with one or more of the following symptoms: intimidating behaviour, aggression to property, demeaning or hostile verbal behaviour, or aggression to persons; (iii) Agitation was defined according to Lindenmayer  as a staPatients' antipsychotic treatment was categorised as including any olanzapine or not (Olz or non-Olz), including any risperidone or not (Ris or non-Ris), and including any haloperidol or not . The Olz, Ris, and Hal cohorts thus overlap, because each cohort included all patients who received the respective drug in any amount and at any time throughout the 5-day study period.Benzodiazepine dosages are presented as cumulative dose during the complete study period, expressed as mg diazepam equivalents. Those were calculated based on the data published by Poser and Poser in 1996 [The following instruments were completed at each visit: Agitation was measured using the Excited Component of the Positive and Negative Syndrome Scale (PANSS) ,11. The Based on market research data for the clinical setting from January 2002 through January 2003, it was expected that cohorts would contain at least 75 patients and up to approximately 300 patients. Based on clinical trial data it was assumed that 43% to 53% of patients would receive benzodiazepine co-medication, and the corresponding two-sided 95% confidence intervals for these rates would be approximately 11.2% (N = 75) and 5.6% (N = 300). The standard deviations (SD) of mean daily benzodiazepine doses ranged from 3.2 mg to 5.8 mg in clinical trial data. However, compared to clinical trials, a larger standard deviation of daily benzodiazepine doses must be assumed due to the naturalistic setting of the present study. Assuming a SD of 7 mg leads to two-sided 95% confidence intervals with a distance from mean to limit of 1.58 mg (N = 75) and 0.79 mg (N = 300). Therefore, the planned total number of 500 patients was considered sufficient to observe different therapeutic responses in a population of impulsive/aggressive patients with psychiatric emergencies.Due to the naturalistic and observational design of the study, a specific primary outcome measure as required for an interventional clinical trial was not defined; all analyses were exploratory only. No confirmatory statistical tests were performed, but 95% confidence intervals and p values were calculated for exploratory treatment group comparisons. Chi-square tests were used for categorical data, t-tests with pooled variance were used for continuous data. The odds ratios and the respective confidence intervals for the use of benzodiazepines at any time were calculated by cohort using the logistic regression technique. Except for the PANSS-EC, all parameters were analyzed on a categorical basis whereby each item at each time point was analyzed descriptively.A total of 558 patients with acute behavioural emergencies were enrolled at 102 participating centres (median 5 patients per centre). Patient demographics are presented in Table Oral antipsychotic medication was used in the majority of cases, but parenteral (intravenous or intramuscular) acute or depot formulations were used as well. In the haloperidol group (n = 132), 46 patients (34.9%) received intravenous, 16 patients (12.1%) intramuscular and 7 patients (5.3%) depot injections. In the olanzapine group (n = 389 with data available), 27 patients (6.9%) received an intramuscular preparation. In the risperidone group (n = 72), 5 patients (6.9%) each received intramuscular and depot injections.Most patients were diagnosed with schizophrenia spectrum disorders . Other diagnoses and the various types of psychiatric emergencies are presented in Table Almost all patients completed the 5 days study period (97.4%); 1.2% of patients were lost to follow-up, 0.6% discontinued due to adverse events, and 0.8% discontinued due to other reasons.Overall, 38.4% of patients had received prior antipsychotic treatment Table . PatientDuring the study, the proportion of patients receiving antipsychotic monotherapy was significantly higher in the olanzapine than in the non-olanzapine cohort , but significantly lower in the risperidone vs. the non-risperidone and in the haloperidol vs. non-haloperidol cohorts  or diazepam . Table Overall, 389 patients (69.7%) received concomitant benzodiazepine treatment, mostly lorazepam n = 48, 44.4%Figure The percentage of patients classified as responders (PANSS-EC ≥ 40% reduced) did not differ significantly between treatment cohorts .The proportion of patients who had at least a moderate level of aggression (CGI-A score ≥ 3) decreased progressively over the 5-day observation period, with no marked differences between treatment groups Figure . HoweverThe proportion of patients rated at least moderately suicidal (CGI-SS score ≥ 3) also decreased over the 5-day observation period in all treatment cohorts  decreased from baseline to day 2 Figure . HoweverTreatment-emergent adverse events were reported in 37 patients (6.6%) of patients overall, the overall adverse event rate was in the same order of magnitude for all treatment cohorts , bipolaDue to this urgency, combined with only scarce evidence from controlled clinical studies, treating physicians generally rely upon older established medications  that may differ from more recently-introduced medications such as the atypical APs. Some controlled trials have evaluated typical APs for emergency treatment, underlining their effectiveness. In one controlled, double-blind study, an intramuscular injection of haloperidol and lorazepam was more rapid in achieving emergency tranquilisation than either haloperidol or lorazepam alone . Two larAnother reason for the paucity of controlled clinical trials in patients with acute aggressive or impulsive behaviour is that until recently most atypical APs were not available as intramuscular formulations , which mThe current IMPULSE study investigated treatment of acutely agitated patients including the short-term effectiveness and tolerability of olanzapine and other antipsychotic treatments . The observational nature of this study enabled the inclusion of a highly agitated, heterogeneous group of patients, many of whom would have normally been excluded from randomised trials with strict in- and exclusion criteria. Thus, the results of this study are more representative of those patients generally encountered in an emergency treatment situation. However, as for all observational studies, the lack of randomisation and of independent, blinded assessments are clear limitations of this study. For this study in particular, it needs to be considered that patients receiving olanzapine treatment were over-represented in our population compared to the olanzapine market share, limiting the generalisability and representativity of our results. Furthermore, treatment groups were not fully comparable due to the lack of randomisation. Patients in the olanzapine cohort were less frequently pre-treated with antipsychotics than patients receiving no olanzapine, while patients receiving risperidone were more frequently pre-treated. Also, the olanzapine cohort tended to have lower CGI-A and CGI-SS ratings at baseline. These differences indicate that the olanzapine cohort may represent somewhat less severe patients at baseline.Patients' psychiatric diagnoses and other background characteristics were quite varied, and should be reflecting the wider population of patients requiring immediate treatment for aggression: mean patient age was 40 years, about 63% were male, and large percentages abused psychoactive substances such as nicotine, alcohol, and/or illicit drugs. Large percentages of these patients were considered to be self-endangering or endangering to others, showed psychomotor agitation, or their behaviour required compulsory admission to a hospital or clinic. A majority of the patients in this study had an ICD-10 diagnosis of schizophrenia (F20–F29), and large proportions had mental disorders due to psychoactive substance abuse (F10–F19), mood disorders (F30–F39), or adult personality disorders (F60–F69).About 70% of the patients  in this study received at least one dose of olanzapine during the 5-day treatment and observation period, usually in combination with other APs. The remaining 30% were treated with APs other than olanzapine. The proportion of patients receiving antipsychotic polytherapy was much lower in the olanzapine than in the risperidone or haloperidol cohorts. However, these data may have been biased by the observational and non-randomised study design and the associated over-representation of olanzapine-treated patients.Overall, about 70% of patients did receive benzodiazepines in addition to the AP medications. However, benzodiazepine use was numerically lower in the olanzapine-treated patients than in those receiving risperidone or haloperidol, and daily benzodiazepine doses were numerically lower in patients receiving haloperidol or olanzapine than in those receiving risperidone.For the analyses used in this study, all treatment groups showed clear improvements of effectiveness measures even within the short observation time of this study. The PANSS-EC and CGI-aggression and CGI-suicidality scores improved to a comparable extent in the olanzapine, risperidone and haloperidol treatment groups. Nearly all patients were alert and active at the baseline measurement of the tranquilisation score. Patients in the olanzapine cohort were less sedated than the risperidone or haloperidol cohorts on all post-baseline days. This difference was most pronounced on day 2. This positive effect observed in the olanzapine cohort may have been caused by less frequent use of concomitant benzodiazepines, suggesting that olanzapine may achieve an effective immediate control of aggression without severe tranquilisation of the patient. Intake of lower amounts of benzodiazepines can be expected to reduce the incidence of adverse effects associated with benzodiazepines , and reduce the incidence of adverse events caused by interactions between benzodiazepines and other antipsychotic medications. However, the limitations due to the observational and non-randomised study design need to be considered when evaluating these results.Immediate control of aggressive and impulsive behaviour can be effectively achieved through administration of antipsychotic medications, which are often administered without knowing the definite diagnosis in this specific setting. During the course of this short-term study, improvements in CGI-A and PANSS-EC were similar for olanzapine, risperidone and haloperidol treatment. The odds for receiving antipsychotic monotherapy were higher in patients treated with olanzapine. Compared to patients treated exclusively with other APs, those receiving olanzapine as antipsychotic monotherapy or part of combination treatment experienced a numerical reduction  of benzodiazepine use and a positive effect on tranquilisation in the present non-randomised, naturalistic study. In patients receiving haloperidol, concomitant benzodiazepine use was significantly more frequent than in patients not treated with haloperidol. Given the huge number of patients requiring emergency inpatient or outpatient treatment for aggressive or impulsive behaviour, the use of atypical antipsychotics such as olanzapine may have the potential to reduce the medical burden for patients with this psychiatric condition.AP: antipsychotic; CGI: Clinical Global Impression; CGI-A: CGI-aggression; CGI-SS: CGI-suicidality; EPS: extrapyramidal symptoms; Hal: haloperidol; ICD-10: International Classification of Diseases; LOCF: last observation carried forward; MedDRA: Medical Dictionary for Regulatory Activities; Olz: olanzapine; PANSS: Positive and Negative Syndrome Scale; Ris: risperidone.The study was funded and conducted by Lilly Deutschland GmbH, Bad Homburg, Germany. SW, AS, and TW are current employees of Eli Lilly and Company or Lilly Deutschland GmbH.SW, AS and TW participated in the design of the study and data interpretation and revised the manuscript. TW participated in the design of the study, prepared the protocol, participated in data interpretation, drafted the final study report, and reviewed the manuscript. AS provided statistical advice, participated in the statistical analyses and reviewed the manuscript. SW participated in data interpretation and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium falciparum malaria by Freya Fowkes and colleagues aids identification of antigens that confer protection from malaria.A systematic review and meta-analysis examining the association between anti-merozoite antibody responses and incidence of  One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting.Plasmodium falciparum malaria. Two independent researchers searched six databases and identified 33 studies that met predefined inclusion and quality criteria, including a rigorous definition of symptomatic malaria. We found that only five studies were performed outside sub-Saharan Africa and that there was a deficiency in studies investigating antibodies to leading vaccine candidates merozoite surface protein (MSP)-142 and erythrocyte binding antigen (EBA)-175. Meta-analyses of most-studied antigens were conducted to obtain summary estimates of the association between antibodies and incidence of P. falciparum malaria. The largest effect was observed with IgG to MSP-3 C terminus and MSP-119  and there was evidence of a dose-response relationship. A tendency towards protective risk ratios (RR<1) was also observed for individual study estimates for apical membrane antigen (AMA)-1 and glutamate-rich protein (GLURP)-R0. Pooled estimates showed limited evidence of a protective effect for antibodies to MSP-1 N-terminal regions or MSP-1-EGF . There was no significant evidence for the protective effect for MSP-2 . Heterogeneity, in terms of clinical and methodological diversity between studies, was an important issue in the meta-analysis of IgG responses to merozoite antigens.We performed a systematic review with meta-analysis of prospective cohort studies examining the association between anti-merozoite immunoglobin (Ig) G responses and incidence of These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans, and to help identify antigens that confer protection from malaria. Further prospective cohort studies that include a larger number of lead antigens and populations outside Africa are greatly needed to ensure generalizability of results. The reporting of results needs to be standardized to maximize comparability of studies. We therefore propose a set of guidelines to facilitate the uniform reporting of malaria immuno-epidemiology observational studies.Please see later in the article for the Editors' Summary Plasmodium falciparum malaria, a mosquito-borne parasitic infection, kills about one million people every year. Around a week after an infected mosquito has bitten a person, “merozoites” (one of the life-stages of the parasite) infect the person's red blood cells where they replicate and then burst out and infect more red blood cells. Rapid replication of parasites can occur in the bloodstream, leading to massive numbers of parasites that can damage vital organs. Although individuals can lower their risk of becoming infected with malaria parasites by avoiding mosquito bites, a vaccine is urgently needed to reduce the global burden of malaria. When malaria parasites infect a person for the first time, the human immune system begins to produce antibodies, proteins that recognize molecules (antigens) on the parasite's surface and that act directly or cooperate with other parts of the immune system to kill malaria parasites. The production of these “naturally acquired” antibodies is initially slow so the individual can become ill when infected. However, because the immune system “remembers” how to make the antibodies, its response to subsequent infections is quicker. The levels of these antibodies also build up with each infection and become more effective at killing parasites. Vaccines, which contain malaria antigens, “prime” the immune system to respond rapidly to malaria infections and produce high concentrations of antibodies to prevent the infection from causing serious illness.P. falciparum malaria in naturally exposed populations in different regions of the world.A malaria vaccine that stimulates an efficient immune response against merozoites would limit the severity of malarial infections and prevent many deaths but no one knows which (if any) of the antigens on merozoites stimulate a protective immune response. Although many different types of antibodies are produced by the immune system, only some of these are effective in protecting against malaria. By investigating whether there is an association between naturally acquired antibodies, which recognize specific candidate antigens, and protection from malaria in populations living in areas where malaria is endemic , vaccine developers can get an idea about which antigens to include in their vaccines. Although many of these “malaria immuno-epidemiological studies” have been undertaken, their results are somewhat conflicting. In this study, the researchers reanalyze these results by doing a systematic review  and a meta-analysis . The researchers evaluated studies of the relationship between anti-merozoite antibodies and the incidence (the number of new cases of a disease in a population per year) of 42, a leading vaccine candidate). Conversely, the association between malaria incidence and antibodies to the antigen MSP-119, which has been included in only one candidate vaccine, was frequently studied. In their meta-analyses, the researchers found that among people with antibodies to the merozoite antigens MSP-3  and MSP-119, the risk of developing P. falciparum malaria was reduced by 54% and 18%, respectively, compared to people without antibodies to these antigens. There was also some evidence of a reduced risk of malaria for people with antibodies to AMA1 and GLURP. For other merozoite antigens, MSP1  and MSP2, there was either weak or no evidence for a protective effect of naturally acquired antibodies.The researchers' search of the published literature yielded 33 studies in which the incidence of malaria had been recorded over time in groups of people in whom levels of antibodies to specific merozoite antigens had been measured. These studies measured antibodies at the start of the study and examined the subsequent risk of malaria over several months of follow-up (these are known as prospective cohort studies). All but five of the studies were performed in Africa, and very few merozoite antigens had been well-studied in different populations, or studied at all. Of note, very few studies had examined naturally acquired antibodies to some leading vaccine candidates  and on the development of malaria vaccinesInformation is available from the malaria (in English and Spanish)The US Centers for Disease Control and Prevention provides information on Wellcome Trust on all aspects of malaria, including vaccine developmentInformation is available from the Malaria Vaccine Initiative provides information on the development of malaria vaccines and on ongoing trialsThe malaria (in English and Spanish)MedlinePlus provides links to additional information on  Plasmodium falciparum is a leading cause of mortality and morbidity globally, particularly among young children. After repeated exposure, individuals develop effective immunity that controls blood-stage parasitaemia, thereby reducing clinical symptoms and life-threatening complications  guidelines for the conduct of meta-analyses of observational studies P. falciparum malaria. Key words included: MSP, merozoite surface protein, MSA, merozoite surface antigen, GLURP, glutamate-rich protein, serine repeat antigen, SERA, S-Antigen, ABRA, AMA, apical membrane antigen, EBA, erythrocyte binding antigen, rhoptry, malaria, P. falciparum, immunity, antibodies, IgG, cohort, longitudinal, incidence, risk, epidemiology, vaccine. The key words variant surface antigen (VSA) were also used because merozoite antigens are sometimes used as comparative antigens in studies investigating VSAs. The reference lists of obtained papers were searched for further studies. Studies reported in languages other than English were included.PubMed, Web of Science, Scopus, Google Scholar, African Index Medicus, and LILACS   were searched for studies examining the association of antibody responses to merozoite antigens with The criteria for inclusion of studies were population-based prospective studies and population-based treatment to reinfection studies. Population-based cross-sectional studies to determine prevalence were excluded because causality cannot be established. Case-control studies, hospital-based studies, and vaccine efficacy trials of blood-stage vaccines were also excluded because of the rigorous inclusion and exclusion criteria applied during these studies, such that the participants would not be representative of the general population.P. falciparum status and studies investigating returned travellers or transmigrants were also excluded as they would not be representative of the general population.The criterion for inclusion of participants was individuals living in malaria endemic areas. Studies restricted to pregnant women and/or children <1 y  were excluded to remove the confounding effect of maternal transferred immunity. Studies where individuals were selected according to their Total immunoglobulin G (IgG) responses to recombinant or synthetic defined merozoite antigens measured at baseline  were considered. IgG responses to full length proteins, processing products, and defined regions of merozoite antigens were included, but IgG responses to peptides that represent undefined regions or incomplete domains or subdomains of antigens were excluded.P. falciparum infection , symptomatic P. falciparum malaria, severe P. falciparum malaria, and P. falciparum malaria-associated mortality. In treatment-to-reinfection studies P. falciparum reinfection was also included as an outcome. Newly established blood-stage infection must have been differentiated from treatment failure by either PCR or documented clearance of infection within a specified time frame appropriate for the chosen antimalarial.The following malaria outcome measures during follow-up were included: high density The minimum quality criteria for inclusion in the review were that: detection of malaria was by active case detection (ACD) and/or passive case detection (PCD); parasitaemia was confirmed by slide microscopy, rapid detection kit, or PCR; symptomatic malaria was defined as fever and/or history of fever (within the past 72 h) plus a high density parasitaemia threshold (to increase specificity because low-grade parasitaemia is common in most settings); severe malaria was defined by the World Health Organization criteria and other causes of morbidity excluded; and other common causes of mortality excluded before a diagnosis of malaria-associated mortality Review authors  identified possible studies, FJIF and JSR assessed the methodological quality of included studies independently, with discrepancies resolved by discussion with JGB.P. falciparum parasitaemia of any density  were invited to provide estimates or data meeting the quality criteria. Some studies had analysed antibody levels at baseline as the outcome variable, comparing baseline levels in those who had or did not have P. falciparum malaria during follow-up. For these studies, data was extracted and reanalysed so that malaria was the outcome variable and related to antibodies at baseline. If the raw data were not presented, authors of the study were invited to reanalyse or provide data for the inclusion of their study in the systematic review. In addition, we contacted several authors whose studies did not meet the inclusion criteria yet contained data that were eligible for the systematic review. These were the authors of studies that had measured antibody responses after baseline  and invited them to provide estimates or data concerning prospective P. falciparum incidence only. We also contacted authors who had restricted analysis to individuals who were parasite positive at baseline and invited them to provide estimates or data on the whole cohort where possible. If authors were unable to provide estimates or data, the study was classified as not meeting inclusion and/or quality criteria and excluded from the systematic review.Authors of studies that had defined a case of symptomatic malaria as fever and/or history of fever plus a Measures of association  and their 95% confidence intervals (CIs) were extracted or derived using data reported in the publications. Data extraction was performed independently by FJIF and JSR, using proforma designed by FJIF, JAS, and JGB. The investigators of the original studies were contacted if relevant information on eligibility or key study data were not available in the published report. An email was sent to authors explaining the nature of the systematic review and the information required together with proforma. If the author did not respond within three email attempts then no further action was taken. Where a study does not provide measures of association , the study results will be described only in narrative terms.A major issue in reviewing the published results of different epidemiologic studies examining the relation between an exposure variable and risk of the outcome is that the results are presented in many different ways. Determining antibody levels by enzyme-linked immunosorbent assay (ELISA) does not produce a common metric measurement among studies. Individuals can be classified as “responders” or “nonresponders” relative to a negative control (unexposed sera) within each study. Study-specific comparisons of these exposure variables can then be pooled. However, categories based on arbitrary cut-offs  cannot be pooled across studies.For studies where the antibody measures were analysed as continuous exposure variables we either asked the authors to reanalyse their data by collapsing the antibody data into categories or asked them to provide the standard deviation of the data so we could calibrate the estimate to represent the relative change in the risk of malaria associated with a change of one standard deviation of the antibody level. For log transformed antibody data we used log base 2 so that the relative change in malaria risk corresponds to a doubling of antibody level.P. falciparum parasitaemia (at baseline or preseason), and/or bed net use are reported. Estimates adjusted for other anti-merozoite antibodies (including antibodies to schizont protein extract) are not reported because antibody responses are typically highly correlated making it difficult to estimate their individual regression coefficients reliably; in these cases unadjusted estimates are reported. For all malaria outcomes the study-assigned P. falciparum definitions were used.ORs considerably overestimate the RR, if the incidence risk is >20%, which is often the case in highly malaria endemic areas 42, MSP-119 ). Estimates from the above-mentioned regions/alleles were used to ensure maximum comparability between studies. Separate analyses were not done for MSP-3-Ct or MSP-119 alleles because of the conserved nature of MSP-3-Ct and MSP-119 (similarly EGF domains). For these antigens, if responses to multiple alleles were investigated in the same study, the most common circulating allele in the population was included in the meta-analysis.Our aim was to obtain a single RR estimate for each study. If antibody responses to the same antigen, in the same population-based study, were reported in several publications, results from the largest sample size were used. Separate estimates were obtained for the RR associated with AMA-1 (full-length ectodomains of FVO [pro-DI-DII-DIII] and 3D7 [DI-DII-DIII]), EBA-175 , GLURP , MSP-2 , MSP-3 . For MSP-1, separate analyses were done for each region and allelic type , MAD20-like [MAD20], and RO33-like [RO33]), and processing fragments  of the RR was calculated using the formula (ln[upper limit of CI]−ln[RR])/1.96. Heterogeneity between studies was tested with the Clinical and methodological heterogeneity was explored using prespecified variables to minimize spurious findings. Variables evaluated included study design , length of follow-up, age of study participants , malaria endemicity , source of malaria cases , definition of symptomatic malaria, preparation of antigen , and method of antibody determination . Influence analysis was also performed whereby pooled estimates were calculated omitting one study at a time. Where possible, publication bias was assessed visually by plotting a funnel plot P. falciparum malaria was examined in 19, eight, seven, five, three, and six studies, respectively. Details of recombinant proteins and sero-prevalences can be found in P. falciparum malaria during follow-up was the most common outcome, examined in 29 studies; with reinfection and high density infection during follow-up examined in five and three studies, respectively. No study examined the association of anti-merozoite responses with incidence risk of severe P. falciparum malaria or P. falciparum malaria-associated mortality.The 33 studies reported data obtained from 14 separate prospective cohort studies and six separate treatment-to-reinfection population studies (42) fragments 42 is further processed into MSP-119 and MSP-133 fragments. Both MSP-119 and MSP-142 are regarded as potential vaccine candidates and have been shown to be protective in animal models 19 IgG responders had an 18% reduction in the risk of symptomatic P. falciparum malaria compared to nonresponders  with the greatest magnitude of effect seen with MAD20 and Palo Alto alleles  that is proteolytically processed into 83 kDa, 30 kDa, 38 kDa, and C-terminal 42 kDa (MSP-119 levels (log base 2) and the risk of malaria P. falciparum per doubling of antibody levels was observed  19 responses with P. falciparum high density infection or reinfection . Meta-analyses showed no association between the presence of responses to either MSP-1-EGF1 or MSP-1-EGF2 with protection against symptomatic P. falciparum  MSP-142 levels (log base 2) with incidence risk of symptomatic P. falciparum and found a reduced risk  Only one study examined the association of MSP-1P. falciparum with allelic specific MSP-1 block 2 responders compared to nonresponders was examined in four studies p = 0.39) or RO33-like allele   . There wP. falciparump = 0.031) but not MSP-1 block 2 MAD20-like repeats   and incidence risk of symptomatic  = 0.35) . There wp = 0.88; Combined results from two studies showed no evidence of an association of MSP-1 block 1 responses with risk of symptomatic falciparum malaria  and risk of symptomatic P. falciparum  but not MSP-2FC27  P. falciparum  The single P. falciparum in responders versus nonresponders  is highly conserved whereas the remainder of the sequence is defined by two major allelic types, 3D7 and K1 3D7 and MSP-3K1 responses (responders versus nonresponders) with risk of symptomatic P. falciparum3D7 responses . A high degree of heterogeneity was also seen for MSP-3K1 associations  so results were not combined , and there was also a tendency towards a protective effect in the study that examined tertiles , but combined results of two studies showed no association of anti-AMA-1FVO levels (log base 2) with incidence risk of symptomatic P. falciparum  tertiles  [40],,[74Text S1PRISMA checklist.(0.07 MB DOC)Click here for additional data file.Text S2Excluded studies, supplementary tables, and analyses.(0.54 MB DOC)Click here for additional data file.


An outbreak of avian influenza (H7N3) among poultry resulted in laboratory-confirmed disease in 1 of 103 exposed persons. Incomplete use of personal protective equipment (PPE) was associated with conjunctivitis and influenza-like symptoms. Rigorous use of PPE by persons managing avian influenza outbreaks may reduce exposure to potentially hazardous infected poultry materials. In April 2006, an outbreak of avian influenza occurred on 3 poultry farms in Norfolk, England  infection. We pretested and then administered a questionnaire by telephone after poultry culling ended . For persons who did not respond to the questionnaire (n = 39), we extracted data recorded in the outbreak records to describe their activities in relation to the outbreak, their use of oseltamivir prophylaxis, and their seasonal influenza vaccine status. Only persons who were interviewed and completed the questionnaire (n = 103) were included in the statistical analysis. Persons were invited to provide an acute-phase blood sample during the outbreak and a convalescent-phase sample 28 days after their last potential exposure. Exceptions were those at low risk; e.g., incinerator workers and lorry drivers.>1 of the following: fever, sore throat, cough, shortness of breath, body/muscle pain, runny nose) in the 7 days after last potential exposure. Confirmed case-patients were those for whom virus was detected by culture and RT-PCR of material from the conjunctiva or respiratory tract and/or confirmed by serologic testing. Influenza virus (H7N3) from the conjunctiva of the index case-patient was prepared by growth in embryonated eggs. Serum samples were screened by using microneutralization (MN) and hemagglutination inhibition (HI) tests  were retained regardless of p value.We calculated odds ratios (ORs), 95% confidence intervals (CIs), and p values for being a possible or confirmed case-patient. Independent variables are shown in the In total, 142 persons were potentially exposed. Questionnaires were completed for 103 (73%) persons . Characteristics, potential exposures, and preventive measures differed little between persons who did or did not complete the questionnaire . Of 46 pFifty-six (54%) persons reported complete use of PPE. Single-variable analysis indicated that working on an infected premise  was significantly associated with being a possible or confirmed case-patient , Table ASerum samples were available from 91 persons: 33 acute- and convalescent-phase pairs, 49 acute-phase samples, and 9 convalescent-phase samples. Only the serum from the index case-patient showed reactivity in both the MN (titer 40) and HI (titer 32) tests and also showed reactivity in Western blot. No acute-phase sample from this person was available. All other acute- and convalescent-phase samples were negative in both tests. During the outbreak, eye, nose, and throat swabs were taken from 14 persons (1–8 days after symptom onset); 10 reported influenza-like symptoms (2 without eye involvement), 2 reported no symptoms, and 2 had no clinical information available. Comprehensive molecular diagnostic tests for common human viral respiratory pathogens  were also performed and did not provide evidence of alternative causes of infection. A vaccine strain of avian paramyxovirus (Newcastle disease virus) was recovered from 1 person with conjunctivitis, which suggests that at least 1 case of conjunctivitis was caused by avian paramyxovirus. Serologic testing for seasonal influenza infection  did not indicate any recent human infections.Our study had a number of limitations. Because workers were interviewed a minimum of 2 months after the outbreak, they may not have accurately recalled their exposures. In addition, we relied on self-reported data. Difficulties recalling symptoms were less likely as we actively followed up persons for 7 days after last exposure. In the absence of a control group, such as farmers from noninfected premises, whether the incidence of influenza-like illness and conjunctivitis in this cohort was different is unclear, although during the outbreak, influenza activity in the general population was low and no isolates of seasonal influenza were reported. We did not measure dust exposure as an alternative explanation for conjunctivitis in some or all persons, apart from the index case-patient who reported this symptom. The results from laboratory testing were limited because convalescent-phase serum was not available from all persons who reported influenza-like illness. However, a wide range of molecular diagnostic tests for human viral pathogens were performed on samples from persons who were not well at the time of the outbreak. Because the kinetics of appearance and disappearance of human antibodies to avian influenza are poorly understood, timing of the collection of samples may not have been optimal in this outbreak and we may have missed the opportunity to diagnose some infections. Moreover, because serologic tests for influenza virus A (H7N3) may not correlate well with infection


From a high-quality population-based register of children with cancer, 455 cases diagnosed with central nervous system (CNS) tumours were analysed to examine patterns of occurrence and geographical distribution. There was a significant increase of 1.8%  in average annual incidence for all CNS tumours, mainly accounted for by a 3.1% rise  in primitive neuroectodermal tumours (PNETs) over the 22-year period 1974-95. These increases were not explained by an increase in the proportion of histologically verified tumours. In the most recent time period (1986-95), astrocytomas occurred more commonly than previously in 0 to 4-year olds. Geographical differences in incidence were evident at a large scale, between counties, for all tumours and astrocytomas, with lower rates in the most urbanized areas. At the level of census district and electoral wards, no association between incidence of CNS tumours and socioeconomic group, person-based population density or ethnicity was observed using Poisson regression modelling. Based on small-scale census geography, the patterns of distribution of CNS tumours do not suggest strong associations with geographical determinants of risk. This study finds a rising incidence of all CNS tumours and particularly primitive neuroectodermal tumours and shows that astrocytomas appear to be occurring at a younger age, most probably because of improved diagnosis with non-invasive technology.