Specific proteome changes in platelets from individuals with GATA1-, GFI1B-, and RUNX1-linked bleeding disorders.

Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Evaluation of a standardised protocol to measure the disease burden of respiratory syncytial virus infection in young children in primary care.

BACKGROUND: A better understanding of the burden of respiratory syncytial virus (RSV) infections in primary care is needed for policymakers to make informed decisions regarding new preventive measures and treatments. The aim of this study was to develop and evaluate a protocol for the standardised measurement of the disease burden of RSV infection in primary care in children aged < 5 years. METHODS: The standardised protocol was evaluated in Italy and the Netherlands during the 2019/20 winter. Children aged < 5 years who consulted their primary care physician, met the WHO acute respiratory infections (ARI) case definition, and had a laboratory confirmed positive test for RSV (RT-PCR) were included. RSV symptoms were collected at the time of swabbing. Health care use, duration of symptoms and socio-economic impact was measured 14 days after swabbing. Health related Quality of life (HRQoL) was measured using the parent-proxy report of the PedsQL™4.0 generic core scales (2-4 years) and PedsQL™4.0 infant scales (0-2 years) 30 days after swabbing. The standardised protocol was evaluated in terms of the feasibility of patient recruitment, data collection procedures and whether parents understood the questions. RESULTS: Children were recruited via a network of paediatricians in Italy and a sentinel influenza surveillance network of general practitioners in the Netherlands. In Italy and the Netherlands, 293 and 152 children were swabbed respectively, 119 and 32 tested RSV positive; for 119 and 12 children the Day-14 questionnaire was completed and for 116 and 11 the Day-30 questionnaire. In Italy, 33% of the children had persistent symptoms after 14 days and in the Netherlands this figure was 67%. Parents had no problems completing questions concerning health care use, duration of symptoms and socio-economic impact, however, they had some difficulties scoring the HRQoL of their young children. CONCLUSION: RSV symptoms are common after 14 days, and therefore, measuring disease burden outcomes like health care use, duration of symptoms, and socio-economic impact is also recommended at Day-30. The standardised protocol is suitable to measure the clinical and socio-economic disease burden of RSV in young children in primary care.

Differential effects of long-term aerobic versus cognitively-engaging physical activity on children's visuospatial working memory related brain activation: A cluster RCT.

Different types of physical activity are thought to differentially affect children's brain activation, via physiological mechanisms, or by activating similar brain areas during physical and cognitive tasks. Despite many behavioral studies relying on these mechanisms, they have been rarely studied. This study looks at both mechanisms simultaneously, by examining effects of two physical activity interventions (aerobic vs. cognitively-engaging) on children's brain activation. Functional Magnetic Resonance Imaging (fMRI) data of 62 children (48.4% boys, mean age 9.2 years) was analyzed. Children's visuospatial working memory related brain activity patterns were tested using a Spatial Span Task before and after the 14-week interventions consisting of four physical education lessons per week. The control group followed their regular program of two lessons per week. Analyses of activation patterns in SPM 12.0 revealed no activation changes between pretest and posttest (p > .05), and no differences between the three conditions in pretest-posttest changes in brain activation (p > .05). Large inter-individual differences were found, suggesting that not every child benefited from the interventions in the same way. To get more insight into the assumed mechanisms, further research is needed to understand whether, when, for whom, and how physical activity results in changed brain activation patterns.

Relationships between gross motor skills, cardiovascular fitness, and visuospatial working memory-related brain activation in 8- to 10-year-old children.

Relationships between gross motor skills and cardiovascular fitness with visuospatial working memory (VSWM) in children are hypothesized to be mediated by underlying functional brain mechanisms. Because there is little experimental evidence to support this mechanism, the present study was designed to investigate the relationships of gross motor skills and cardiovascular fitness with VSWM-related brain activation in 8- to 10-year-old children. Functional magnetic resonance imaging data obtained during a VSWM-task were analyzed for 80 children from grades 3 (47.5%) and 4 of 21 primary schools in the Netherlands (51.3% girls). Gross motor skills (Korper Koordinationstest für Kinder and Bruininks-Oseretsky Test of Motor Proficiency - 2nd Edition) and cardiovascular fitness (20-meter Shuttle Run Test) were assessed. VSWM-related brain activation was found in a network involving the angular gyrus, the superior parietal cortex, and the thalamus; deactivation was found in the inferior and middle temporal gyri. Although behavioral results showed significant relations of gross motor skills and cardiovascular fitness with VSWM performance, gross motor skills and cardiovascular fitness were not related to VSWM-related brain activation. Therefore, we could not confirm the hypothesis that brain activation underlies the relationship of gross motor skills and cardiovascular fitness with VSWM performance. Our results suggest that either the effects of physical activity on cognition do not necessarily go via changes in gross motor skills and/or cardiovascular fitness, or that brain activation patterns as measured with the blood-oxygen-level dependent (BOLD) signal may not be the mechanism underlying the relationships of gross motor skills and cardiovascular fitness with VSWM.

The incidence of symptomatic infection with influenza virus in the Netherlands 2011/2012 through 2016/2017, estimated using Bayesian evidence synthesis.

Due to differences in the circulation of influenza viruses, distribution and antigenic drift of A subtypes and B lineages, and susceptibility to infection in the population, the incidence of symptomatic influenza infection can vary widely between seasons and age-groups. Our goal was to estimate the symptomatic infection incidence in the Netherlands for the six seasons 2011/2012 through 2016/2017, using Bayesian evidence synthesis methodology to combine season-specific sentinel surveillance data on influenza-like illness (ILI), virus detections in sampled ILI cases and data on healthcare-seeking behaviour. Estimated age-aggregated incidence was 6.5 per 1000 persons (95% uncertainty interval (UI): 4.7-9.0) for season 2011/2012, 36.7 (95% UI: 31.2-42.8) for 2012/2013, 9.1 (95% UI: 6.3-12.9) for 2013/2014, 41.1 (95% UI: 35.0-47.7) for 2014/2015, 39.4 (95% UI: 33.4-46.1) for 2015/2016 and 27.8 (95% UI: 22.7-33.7) for season 2016/2017. Incidence varied substantially between age-groups (highest for the age-group <5 years: 23 to 47/1000, but relatively low for 65+ years: 2 to 34/1000 over the six seasons). Integration of all relevant data sources within an evidence synthesis framework has allowed the estimation - with appropriately quantified uncertainty - of the incidence of symptomatic influenza virus infection. These estimates provide valuable insight into the variation in influenza epidemics across seasons, by virus subtype and lineage, and between age-groups.

Catheter ablation of symptomatic idiopathic ventricular arrhythmias : A five-year single-centre experience.

AIMS: This study was designed to gain insight into the patient characteristics, results and possible complications of ablation procedures for symptomatic idiopathic premature ventricular complexes (PVC) and idiopathic ventricular tachycardia (VT). METHODS: Data were collected from all patients who underwent radiofrequency catheter ablation for symptomatic PVCs and idiopathic VT in the Catharina Hospital between 1 January 2011 and 31 December 2015. The procedural endpoint was elimination or non-inducibility of the clinical arrhythmia. Successful sustained ablation was defined as the persistent elimination of at least 80% of the PVCs or the absence of VTs at follow-up. In case of suspected PVC-induced cardiomyopathy, the systolic left ventricular function was reassessed 3 months post procedure. RESULTS: Our cohort consisted of 131 patients who underwent one or more ablation procedures; 99 because of symptomatic premature ventricular complexes, 32 because of idiopathic VT. In total 147 procedures were performed. The procedural ablation success rate was 89%. Successful sustained ablation rate was 82%. Eighteen (13.2%) patients had suspected PVC-induced cardiomyopathy. In 15 of them (83%), successful sustained ablation was achieved and the left ventricular ejection fraction improved from a mean of 39% (±8.8) to 55.4% (±8.1). Most arrhythmias originated from the right ventricular outflow tract (60%) or aortic cusps (13%). Complications included three tamponades. CONCLUSION: Catheter ablation therapy for idiopathic ventricular arrhythmias is very effective with a sustained success rate of 82%. In patients with PVC-induced cardiomyopathy, it leads to improvement of systolic left ventricular function. However, risk for complications is not negligible, even in experienced hands.

Antiviral susceptibility profile of influenza A viruses; keep an eye on immunocompromised patients under prolonged treatment.

There was an increase in severe and fatal influenza cases in Greece during the 2011-2015 post-pandemic period. To investigate causality, we determined neuraminidase (NA) inhibitor susceptibility and resistance-conferring NA and hemagglutinin (HA) mutations in circulating influenza type A viruses during the pandemic (2009-2010) and post-pandemic periods in Greece. One hundred thirty-four influenza A(H1N1)pdm09 and 95 influenza A(H3N2) viruses submitted to the National Influenza Reference Laboratory of Southern Greece were tested for susceptibility to oseltamivir and zanamivir. Antiviral resistance was assessed by neuraminidase sequence analysis, as well as the fluorescence-based 50 % inhibitory concentration (IC50) method. Five influenza A(H1N1)pdm09 viruses (2.2 %) showed significantly reduced inhibition by oseltamivir (average IC50 300.60nM vs. 1.19nM) by Gaussian kernel density plot analysis. These viruses were isolated from immunocompromised patients and harbored the H275Y oseltamivir resistance-conferring NA substitution. All A(H1N1)pdm09 viruses were zanamivir-susceptible, and all A(H3N2) viruses were susceptible to both drugs. Oseltamivir-resistant viruses did not form a distinct cluster by phylogenetic analysis. Permissive mutations were detected in immunogenic and non immunogenic NA regions of both oseltamivir- resistant and susceptible viruses in the post-pandemic seasons. Several amino acid substitutions in the HA1 domain of the HA gene of post-pandemic viruses were identified. This study indicated low resistance to NAIs among tested influenza viruses. Antiviral resistance emerged only in immunocompromised patients under long-term oseltamivir treatment. Sequential sample testing in this vulnerable group of patients is recommended to characterise resistance or reinfection and viral evolution.

Spatial distribution of electrical reconnection after pulmonary vein isolation in patients with recurrent paroxysmal atrial fibrillation.

INTRODUCTION: Recurrence of atrial fibrillation after pulmonary vein isolation (PVI) occurs frequently and may be associated with electrical reconnection of the pulmonary veins (PV). We investigated spatial distribution of electrical reconnection during re-do procedures in patients with paroxysmal atrial fibrillation who had previous successful acute electrical PVI with either single irrigated tip, antral ablation (s-RF; n = 38) or multi-electrode, duty-cycled ablation (PVAC; n = 48). METHODS AND RESULTS: EP navigator, mapping and irrigated tip ablation catheters were used in all re-do procedures. Sites of reconnection were assessed in a 12-segment model. Baseline clinical and demographic characteristics were similar in both groups. The number of PVs reconnected per patient was similar in both groups (2.9 ± 0.9 and 3.2 ± 0.7 (p = 0.193), s­RF and PVAC, respectively), and each PV was equally affected. However, the inferior quadrant of the right lower PV was significantly more vulnerable to reconnection after previous PVAC ablation, whereas the superior quadrant of the right upper PV showed significantly more reconnection in the s­RF group. CONCLUSION: The overall number of PVs reconnected was equally high in both groups, and each PV was affected equally. However, there were significant differences in the spatial distribution of electrical reconnection. Better understanding of predilection sites for reconnection might help to improve the long-term success rate of PVI.

Evaluating hydrogen bonding control in the diastereoselective Diels-Alder reactions of 9-(2-aminoethyl)-anthracene derivatives.

Several 9-(2-aminoethyl)anthracene derivatives were prepared with different nitrogen substitutents including alkyl, acetamide, trifluoroacaeamide and t-butyl carbamate. The selectivity in Diels-Alder cyclodaddition reaction with N-methyl maleimide was evaluated through single crystal X-ray analysis of the products. Models for the change in selectivity with hydrogen bond acceptor are proposed, supported by DFT level calculations.

Age-specific differences in influenza virus type and subtype distribution in the 2012/2013 season in 12 European countries.

The epidemiology of seasonal influenza is influenced by age. During the influenza season, the European Influenza Surveillance Network (EISN) reports weekly virological and syndromic surveillance data [mostly influenza-like illness (ILI)] based on national networks of sentinel primary-care providers. Aggregated numbers by age group are available for ILI, but not linked to the virological data. At the end of the influenza season 2012/2013, all EISN laboratories were invited to submit a subset of their virological data for this season, including information on age. The analysis by age group suggests that the overall distribution of circulating (sub)types may mask substantial differences between age groups. Thus, in cases aged 5-14 years, 75% tested positive for influenza B virus whereas all other age groups had an even distribution of influenza A and B viruses. This means that the intepretation of syndromic surveillance data without age group-specific virological data may be misleading. Surveillance at the European level would benefit from the reporting of age-specific influenza data.

Future directions for the European influenza reference laboratory network in influenza surveillance.

By defining strategic objectives for the network of influenza laboratories that have national influenza centre status or national function within European Union Member States, Iceland and Norway, it is possible to align their priorities in undertaking virological surveillance of influenza. This will help maintain and develop the network to meet and adapt to new challenges over the next 3-5 years and underpin a longer-term strategy over 5-10 years. We analysed the key activities undertaken by influenza reference laboratories in Europe and categorised them into a framework of four key strategic objectives areas: enhancing laboratory capability, ensuring laboratory capacity, providing emergency response and translating laboratory data into information for public health action. We make recommendations on the priority areas for future development.

Aggregation and Association of NDVI, Boll Injury, and Stink Bugs in North Carolina Cotton.

Sampling of herbivorous stink bugs in southeastern U.S. cotton remains problematic. Remote sensing was explored to improve sampling of these pests and associated boll injury. Two adjacent 14.5-ha cotton fields were grid sampled in 2011 and 2012 by collecting stink bug adults and bolls every week during the third, fourth, and fifth weeks of bloom. Satellite remote sensing data were collected during the third week of bloom during both years, and normalized difference vegetation index (NDVI) values were calculated. Stink bugs were spatially aggregated on the third week of bloom in 2011. Boll injury from stink bugs was spatially aggregated during the fourth week of bloom in 2012. The NDVI values were aggregated during both years. There was a positive association and correlation between stink bug numbers and NDVI values, as well as injured bolls and NDVI values, during the third week of bloom in 2011. During the third week of bloom in 2012, NDVI values were negatively correlated with stink bug numbers. During the fourth week of bloom in 2011, stink bug numbers and boll injury were both positively associated and correlated with NDVI values. During the fourth week of bloom in 2012, stink bugs were negatively correlated with NDVI values, and boll injury was negatively associated and correlated with NDVI values. This study suggests the potential of remote sensing as a tool to assist with sampling stink bugs in cotton, although more research is needed using NDVI and other plant measurements to predict stink bug injury.

The effects of sleep extension and sleep hygiene advice on sleep and depressive symptoms in adolescents: a randomized controlled trial.

OBJECTIVE: Sleep problems are common and persistent during adolescence and can have negative effects on adolescents' mood. To date, studies that investigate the effects of sleep extension on adolescents' sleep and depressive symptoms are still lacking. This study aims to investigate the effects of gradual sleep extension combined with sleep hygiene advice in adolescents with chronic sleep reduction on objectively measured sleep, self-reported sleep problems and depressive symptoms. METHODS: Fifty-five adolescents with chronic sleep reduction (mean age: 15.44 years; 85.5% females) were included in the study. Participants were randomly assigned to either a sleep extension group (gradual sleep extension by advancing bedtimes in the evening and receiving sleep hygiene advice) or to a control group (no instruction). Sleep was measured with actigraphy during three weeks, the first week was the baseline week, and the last two weeks were the experimental weeks during which sleep was extended. Other outcome variables were self-reported sleep problems (daytime sleepiness, symptoms of insomnia and circadian rhythm sleep disorder) and depressive symptoms, which were assessed before and after the experimental manipulation. RESULTS: During the third week of the experiment, adolescents in the sleep extension group had earlier bedtimes, earlier sleep onsets, spent more time in bed and slept longer than adolescents in the control group. Their chronic sleep reduction, insomnia symptoms and depressive symptoms diminished significantly. In addition, there was a trend of improved circadian rhythm sleep disorder symptoms and sleep quality. CONCLUSION: Gradual sleep extension combined with sleep hygiene advice seems to have beneficial effects on sleep, self-reported sleep problems and depressive symptoms of adolescents with chronic sleep reduction. Although we cannot distinguish between the effects of sleep extension and sleep hygiene advice, the results suggest that advancing bedtimes can extend sleep and improve depressive symptoms.

Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011-2014.

Enterovirus D68 (EV-D68) continued to circulate in a seasonal pattern in the Netherlands, after the outbreak in 2010. Outpatient EV-D68 cases, mainly in the under 20 and 50­59 years age groups, presented with relatively mild respiratory disease. Hospital-based enterovirus surveillance identified more severe cases, mainly in children under 10 years of age. Dutch partial VP1 genomic region sequences from 2012 through 2014 were distributed over three sublineages similar to EV-D68 from the outbreak in the US in 2014.

Influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1).

Factors that trigger human infection with animal influenza virus progressing into a pandemic are poorly understood. Within a project developing an evidence-based risk assessment framework for influenza viruses in animals, we conducted a review of the literature for evidence of human infection with animal influenza viruses by diagnostic methods used. The review covering Medline, Embase, SciSearch and CabAbstracts yielded 6,955 articles, of which we retained 89; for influenza A(H5N1) and A(H7N9), the official case counts of t he World Health Organization were used. An additional 30 studies were included by scanning the reference lists. Here, we present the findings for confirmed infections with virological evidence. We found reports of 1,419 naturally infected human cases, of which 648 were associated with avian influenza virus (AIV) A(H5N1), 375 with other AIV subtypes, and 396 with swine influenza virus (SIV). Human cases naturally infected with AIV spanned haemagglutinin subtypes H5, H6, H7, H9 and H10. SIV cases were associated with endemic SIV of H1 and H3 subtype descending from North American and Eurasian SIV lineages and various reassortants thereof. Direct exposure to birds or swine was the most likely source of infection for the cases with available information on exposure.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL.

BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2. METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer. RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers. CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Adjusted prognostic association of depression following myocardial infarction with mortality and cardiovascular events: individual patient data meta-analysis.

BACKGROUND: The association between depression after myocardial infarction and increased risk of mortality and cardiac morbidity may be due to cardiac disease severity. AIMS: To combine original data from studies on the association between post-infarction depression and prognosis into one database, and to investigate to what extent such depression predicts prognosis independently of disease severity. METHOD: An individual patient data meta-analysis of studies was conducted using multilevel, multivariable Cox regression analyses. RESULTS: Sixteen studies participated, creating a database of 10 175 post-infarction cases. Hazard ratios for post-infarction depression were 1.32 (95% CI 1.26-1.38, P<0.001) for all-cause mortality and 1.19 (95% CI 1.14-1.24, P<0.001) for cardiovascular events. Hazard ratios adjusted for disease severity were attenuated by 28% and 25% respectively. CONCLUSIONS: The association between depression following myocardial infarction and prognosis is attenuated after adjustment for cardiac disease severity. Still, depression remains independently associated with prognosis, with a 22% increased risk of all-cause mortality and a 13% increased risk of cardiovascular events per standard deviation in depression z-score.

Guiding outbreak management by the use of influenza A(H7Nx) virus sequence analysis.

The recently identified human infections with avian influenza A(H7N9) viruses in China raise important questions regarding possible source and risk to humans. Sequence comparison with an influenza A(H7N7) outbreak in the Netherlands in 2003 and an A(H7N1) epidemic in Italy in 1999­2000 suggests that widespread circulation of A(H7N9) viruses must have occurred in China. The emergence of human adaptation marker PB2 E627K in human A(H7N9) cases parallels that of the fatal A(H7N7) human case in the Netherlands.

The 2009 influenza A (H1N1) pandemic. Management and vaccination strategies in The Netherlands.

Prior to 2009, The Netherlands had prepared itself extensively for a potential pandemic. Multidisciplinary guidelines had been drafted to control transmission and limit adverse outcomes for both a phase of early incidental introduction and for a phase with widespread transmission. The Ministry of Health had ensured a supply and distribution schedule for antivirals and negotiated a contract for vaccine purchases. During the pandemic, existing surveillance was expanded, the established infectious disease response structure was activated, and the previously prepared protocols for communication, diagnostics, use of antivirals, and vaccination implementation were operationalized and implemented. When the pandemic turned out to be less severe than many had anticipated, risk communication and rapid modification of guidelines and communication became a major challenge. Antivirals and pandemic vaccines were reserved for those at high risk for severe outcomes only. Overall, the impact of the pandemic was comparable to the impact of an average seasonal influenza epidemic, but with a shift in (severe) outcomes from the very young and elderly toward young adults. Established prepared protocols enabled timely coordinated responses. In preparing for the worst, sufficient attention must be given to preparing for a mild scenario as well.