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Introduction: The Accelerated Development of Vaccine benefit-risk Collaboration in Europe (ADVANCE) public-private collaboration, aimed to develop and test a system for rapid benefit-risk monitoring of vaccines using healthcare databases in Europe. The objective of this proof-of-concept (POC) study was to test the feasibility of the ADVANCE system to generate incidence rates (IRs) per 1000 person-years and incidence rate ratios (IRRs) for risks associated with whole cell- (wP) and acellular- (aP) pertussis vaccines, occurring in event-specific risk windows in children prior to their pre-school-entry booster. Methods: The study population comprised almost 5.1 million children aged 1 month to

Background Although routine NHS data potentially include all patients, confounding limits their use for causal inference. Methods to minimise confounding in observational studies of implantable devices are required to enable the evaluation of patients with severe systemic morbidity who are excluded from many randomised controlled trials. Objectives Stage 1 – replicate the Total or Partial Knee Arthroplasty Trial (TOPKAT), a surgical randomised controlled trial comparing unicompartmental knee replacement with total knee replacement using propensity score and instrumental variable methods. Stage 2 – compare the risk benefits and cost-effectiveness of unicompartmental knee replacement with total knee replacement surgery in patients with severe systemic morbidity who would have been ineligible for TOPKAT using the validated methods from stage 1. Design This was a cohort study. Setting Data were obtained from the National Joint Registry database and linked to hospital inpatient (Hospital Episode Statistics) and patient-reported outcome data. Participants Stage 1 – people undergoing unicompartmental knee replacement surgery or total knee replacement surgery who met the TOPKAT eligibility criteria. Stage 2 – participants with an American Society of Anesthesiologists grade of ≥ 3. Intervention The patients were exposed to either unicompartmental knee replacement surgery or total knee replacement surgery. Main outcome measures The primary outcome measure was the postoperative Oxford Knee Score. The secondary outcome measures were 90-day postoperative complications (venous thromboembolism, myocardial infarction and prosthetic joint infection) and 5-year revision risk and mortality. The main outcome measures for the health economic analysis were health-related quality of life (EuroQol-5 Dimensions) and NHS hospital costs. Results In stage 1, propensity score stratification and inverse probability weighting replicated the results of TOPKAT. Propensity score adjustment, propensity score matching and instrumental variables did not. Stage 2 included 2256 unicompartmental knee replacement patients and 57,682 total knee replacement patients who had severe comorbidities, of whom 145 and 23,344 had linked Oxford Knee Scores, respectively. A statistically significant but clinically irrelevant difference favouring unicompartmental knee replacement was observed, with a mean postoperative Oxford Knee Score difference of Limitations Although some propensity score methods successfully replicated TOPKAT, unresolved confounding may have affected stage 2. Missing Oxford Knee Scores may have led to information bias. Conclusions Propensity score stratification and inverse probability weighting successfully replicated TOPKAT, implying that some (but not all) propensity score methods can be used to evaluate surgical innovations and implantable medical devices using routine NHS data. Unicompartmental knee replacement was safer and more cost-effective than total knee replacement for patients with severe comorbidity and should be considered the first option for suitable patients. Future work Further research is required to understand the performance of propensity score methods for evaluating surgical innovations and implantable devices. Trial registration This trial is registered as EUPAS17435. Funding This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 66. See the NIHR Journals Library website for further project information.

Interrupted time series (ITS) analysis is being increasingly used in epidemiology. Despite its growing popularity, there is a scarcity of guidance on power and sample size considerations within the ITS framework. Our aim of this study was to assess the statistical power to detect an intervention effect under various real-life ITS scenarios. ITS datasets were created using Monte Carlo simulations to generate cumulative incidence (outcome) values over time. We generated 1,000 datasets per scenario, varying the number of time points, average sample size per time point, average relative reduction post intervention, location of intervention in the time series, and reduction mediated via a 1) slope change and 2) step change. Performance measures included power and percentage bias. We found that sample size per time point had a large impact on power. Even in scenarios with 12 pre-intervention and 12 post-intervention time points with moderate intervention effect sizes, most analyses were underpowered if the sample size per time point was low. We conclude that various factors need to be collectively considered to ensure adequate power for an ITS study. We demonstrate a means of providing insight into underlying sample size requirements in ordinary least squares (OLS) ITS analysis of cumulative incidence measures, based on prespecified parameters and have developed Stata code to estimate this.

The Accelerated Development of VAccine benefit-risk Collaboration in Europe (ADVANCE), a public-private consortium, implemented and tested a distributed network system for the generation of evidence on the benefits-risks of marketed vaccines in Europe. We tested the system by estimating the incidence rate (IR) of pertussis and pertussis-related complications in children vaccinated with acellular (aP) and whole-cell (wP) pertussis vaccine. Data from seven electronic databases from four countries (Denmark: AUH and SSI, Spain: SIDIAP and BIFAP, UK: THIN and RCGP RSC and Italy: Pedianet) were included in a retrospective cohort analysis. Exposure was defined as any pertussis vaccination (aP or wP). The follow-up time started 14 days after the first dose. Children who had received any pertussis vaccine from January 1990 to December 2015 were included (those who switched type, or had unknown type were excluded). The outcomes of interest were confirmed or suspected pertussis and pertussis-related pneumonia and generalised convulsions within one month of pertussis diagnosis and death within three months of pertussis diagnosis. The cohort comprised 2,886,367 children ≤5 years of age. Data on wP and aP vaccination were available in three and seven databases, respectively. The IRs (per 100,000 person-years) for pertussis varied largely and ranged between 0.15 (95% CI: 0.12; 0.19) and 1.15 (95% CI: 1.07; 1.23), and the trends over time was consistent with those observed from national surveillance databases for confirmed pertussis. The pertussis IRs decreased as the number of wP and aP vaccine doses increased. Pertussis-related complications were rare (89 pneumonia, 7 generalised convulsions and no deaths) and their relative risk (vs. non-pertussis) could not be reliably estimated. The study demonstrated the feasibility of the ADVANCE system to estimate the change in pertussis IRs following pertussis vaccination. Larger sample sizes would provide additional power to compare the risk for complications between children with and without pertussis. The feasibility of vaccine-type specific effectiveness studies may be considered in the future.

The Accelerated Development of VAccine beNefit-risk Collaboration in Europe (ADVANCE) is a public–private collaboration aiming to develop and test a system for rapid vaccine benefit-risk monitoring using existing European healthcare databases. Incidence rate (IR) estimates of vaccination-associated adverse events that are needed to model vaccination risks can be calculated from existing healthcare databases when vaccination (exposure) data are available. We assessed different methods to derive IRs in risk periods following vaccination when exposure data are missing in one database, using estimated IRs and IRRs from other databases for febrile seizures, fever and persistent crying. IRs were estimated for children aged 0–5 years in outcome-specific risk and non-risk periods following the first dose of acellular pertussis (aP) vaccination in four primary care databases and one hospital database. We compared derived and observed IRs in each database using three methods: 1) multiplication of non-risk period IR for database i by IR ratio (IRR) obtained from meta-analysis of IRRs estimated using the self-controlled case-series method, from databases other than i; 2) same method as 1, but multiplying with background IR; and 3) meta-analyses of observed IRs from databases other than i. IRs for febrile seizures were lower in primary care databases than the hospital database. The derived IR for febrile seizures using data from primary care databases was lower than that observed in the hospital database, and using data from the hospital database gave a higher derived IR than that observed in the primary care database. For fever and persistent crying the opposite was observed. We demonstrated that missing IRs for a post-vaccination period can be derived but that the type of database and the method of event data capture can have an impact on potential bias. We recommend IRs are derived using data from similar database types (hospital or primary care) with caution as even this can give heterogeneous results.

Background Data on the risk of death following an asthma exacerbation are scarce. With this multinational cohort study, we assessed all-cause mortality rates, mortality rates following an exacerbation, and patient characteristics associated with all-cause mortality in asthma. Methods Asthma patients aged ≥18 years and with ≥1 year of follow-up were identified in 5 European electronic databases from the Netherlands, Italy, UK, Denmark and Spain during the study period January 1, 2008–December 31, 2013. Patients with asthma-COPD overlap were excluded. Severe asthma was defined as use of high dose ICS + use of a second controller. Severe asthma exacerbations were defined as emergency department visits, hospitalizations or systemic corticosteroid use, all for reason of asthma. Results The cohort consisted of 586,436 asthma patients of which 42,611 patients (7.3%) had severe asthma. The age and sex standardized all-cause mortality rates ranged between databases from 5.2 to 9.5/1000 person-years (PY) in asthma, and between 11.3 and 14.8/1000 PY in severe asthma. The all-cause mortality rate in the first week following a severe asthma exacerbation ranged between 14.1 and 59.9/1000 PY. Mortality rates remained high in the first month following a severe asthma exacerbation and decreased thereafter. Higher age, male gender, comorbidity, smoking, and previous severe asthma exacerbations were associated with mortality. Conclusion All-cause mortality following a severe exacerbation is high, especially in the first month following the event. Smoking cessation, comorbidity-management and asthma-treatment focusing on the prevention of exacerbations might reduce associated mortality.

Background Long-term safety and efficacy for everolimus-eluting stents (EES) versus those of sirolimus-eluting stents (SES) are unknown. Objectives This study compared 5-year outcomes for EES with those for SES from the SORT OUT IV (Scandinavian Organization for Randomized Trials with Clinical Outcome) trial. Methods Five-year follow-up was completed for 2,771 patients (99.9%). Primary endpoint was a composite of major adverse cardiac events (MACE), including cardiac death, myocardial infarction (MI), target vessel revascularization (TVR), and definite stent thrombosis. Results At 5-years, MACE occurred in 14.0% and 17.4% in the EES and SES groups, respectively (hazard ratio [HR]: 0.80, 95% confidence interval [CI]: 0.66 to 0.97; p = 0.02). The MACE rate did not differ significantly within the first year (HR: 0.96, 95% CI: 0.71 to 1.19; p = 0.79), but from years 1 through 5, the MACE rate was lower with EES (HR: 0.71, 95% CI: 0.55 to 0.90; p = 0.006; p interaction = 0.12). Definite stent thrombosis was lower with EES (0.4%) than with SES (2.0%; HR: 0.18, 95% CI: 0.07 to 0.46), with a lower risk of very late definite stent thrombosis in the EES group (0.2% vs. 1.4%, respectively; HR: 0.16, 95% CI: 0.05 to 0.53). When censoring the patients at the time of stent thrombosis, we found no significant differences between the 2 stent groups for MACE rates (HR: 0.89, 95% CI: 0.73 to 1.08; p = 0.23), target lesion revascularization (HR: 0.90, 95% CI: 0.64 to 1.27; p = 0.55), and MI (HR: 0.93, 95% CI: 0.64 to 1.36; p = 0.72). Conclusions At 5-year follow-up, MACE rate was significantly lower with EES- than with SES-treated patients, due largely due to a lower risk of very late definite stent thrombosis. (Randomized Clinical Comparison of the Xience V and the Cypher Coronary Stents in Non-selected Patients With Coronary Heart Disease [SORT OUT IV]; NCT00552877 )

The original version of this article, published on 26 November 2019 contained a mistake. An author’s name was misspelled. The correct author name reads as follows: Susana Perez-Gutthann. The original article has been corrected.

Purpose The aim of this article is to provide a detailed description of the ongoing nationwide Danish Centre for Strategic Research in Type 2 Diabetes (DD2) project cohort and biobank. The DD2 cohort continuously enrols newly diagnosed patients with type 2 diabetes (T2D) throughout Denmark. The overall goal of the DD2 project is to establish a large and data-rich T2D cohort that can serve as a platform for exhaustive T2D research including (1) improved genotypic and phenotypic characterisation of T2D, (2) intervention studies of more individualised T2D treatment, (3) pharmacoepidemiological studies and (4) long-term follow-up studies on predictors of T2D complications and prognosis. Participants Between 2010 and 2016, 7011 individuals with T2D have been enrolled and assessed at baseline. Information collected include interview data (eg, body weight at age 20 years, physical activity and alcohol consumption), clinical examination data (eg, hip–waist ratio and resting heart rate) and biological samples (whole blood, DNA, plasma and urine) stored at −80°C and currently analysed for a range of biomarkers and genotypes. Findings to date Registry linkage has provided extensive supplemental continuous data on glycosylated haemoglobin A, lipids, albuminuria, blood pressure, smoking habits, body mass index, primary care contacts, hospital diagnoses and procedures, medication use, cancer and mortality. Cross-sectional associations between biomarkers, family history, anthropometric and lifestyle measures and presence of complications at baseline have been reported. Future plans During 2016, a detailed follow-up questionnaire has been answered by 85% of initial participants, providing follow-up information on baseline variables and on presence of diabetic neuropathy. The DD2 cohort has now been followed for a total of 18 862 person-years, and nested intervention trials and follow-up studies are ongoing. In the future, the cohort will serve as a strong national and international resource for recruiting patients to nested case studies, clinical trials, postmarketing surveillance, large-scale genome studies and follow-up studies of T2D complications.

Background Previous meta-analyses have investigated the relative safety and efficacy profiles of different types of drug-eluting stents (DES) and bare-metal stents (BMS); however, most prior trials in these meta-analyses reported follow-up to only 1 year, and as such, the relative long-term safety and efficacy of these devices are unknown. Many recent studies have now reported extended follow-up data. Objectives This study sought to investigate the long-term safety and efficacy of durable polymer-based DES, bioabsorbable polymer-based biolimus-eluting stents (BES), and BMS by means of network meta-analysis. Methods Randomized controlled trials comparing DES to each other or to BMS were searched through MEDLINE, EMBASE, and Cochrane databases and proceedings of international meetings. Information on study design, inclusion and exclusion criteria, sample characteristics, and clinical outcomes was extracted. Results Fifty-one trials that included a total of 52,158 randomized patients with follow-up duration ≥3 years were analyzed. At a median follow-up of 3.8 years, cobalt-chromium everolimus-eluting stents (EES) were associated with lower rates of mortality, definite stent thrombosis (ST), and myocardial infarction than BMS, paclitaxel-eluting stents (PES), and sirolimus-eluting stents (SES) and less ST than BES. Phosphorylcholine-based zotarolimus-eluting stents had lower rates of definite ST than SES and lower rates of myocardial infarction than BMS and PES. The late rates of target-vessel revascularization were reduced with all DES compared with BMS, with cobalt-chromium EES, platinum chromium-EES, SES, and BES also having lower target-vessel revascularization rates than PES. Conclusions After a median follow-up of 3.8 years, all DES demonstrated superior efficacy compared with BMS. Among DES, second-generation devices have substantially improved long-term safety and efficacy outcomes compared with first-generation devices.

AIMS: To examine the prevalence of micro- and macrovascular complications and their associated clinical characteristics at time of type 2 diabetes (T2D) diagnosis.METHODS: We examined the prevalence of complications and associated clinical characteristics among 6958 newly diagnosed T2D patients enrolled in the prospective Danish Center for Strategic Research in T2D cohort during 2010-2016. We calculated age- and gender-adjusted prevalence ratios (aPRs) of complications using log-binomial and Poisson regression.RESULTS: In total, 35% (n=2456) T2D patients had diabetic complications around diagnosis; 12% (n=828) had microvascular complications, 17% (n=1186) macrovascular complications, and 6% (n=442) had both. HbA1c levels of ≥7% were associated with microvascular complications [HbA1c 7%-8%; aPR: 1.35, 95% confidence interval (CI): 1.12-1.62] but not macrovascular complications [aPR: 0.91, 95% CI: 0.76-1.08]. High C-peptide≥800pmol/L was associated with macrovascular [aPR 1.34, 95% CI: 1.00-1.80] but not microvascular [aPR 0.97, 95% CI: 0.71-1.33] complications. Macrovascular complications were associated with male sex, age>50years, obesity, hypertriglyceridemia, low HDL cholesterol, smoking, elevated CRP levels, and anti-hypertensive therapy. Microvascular complications were associated with high blood pressure, hypertriglyceridemia, and absence of lipid-lowering therapy.CONCLUSIONS: One-third of patients with T2D had diabetes complications around time of diagnosis. Our findings suggest different pathophysiological mechanisms behind micro- and macrovascular complications.

Mathias Ried-Larsen,1–3 Reimar W Thomsen,2,4 Klara Berencsi,4 Cecilie F Brinkløv,1,5 Charlotte Brøns,1,5 Laura S Valentiner,1,6 Kristian Karstoft,1,3 Henning Langberg,1,6 Allan A Vaag,1,2,5 Bente K Pedersen,1,3 Jens S Nielsen7 1Department of Infectious Diseases, Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen, Copenhagen, 2The Danish Diabetes Academy, Odense University Hospital, Odense, 3Department of Infectious Diseases, Centre of Inflammation and Metabolism, Rigshospitalet, University of Copenhagen, Copenhagen, 4Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus Nord, 5Department of Endocrinology (Diabetes and Metabolism), Rigshospitalet, University of Copenhagen, 6CopenRehab, Department of Public Health, Section of Social Medicine, University of Copenhagen, Copenhagen, 7Department of Endocrinology, Odense University Hospital, Odense, Denmark Abstract: Promoting physical activity is a first-line choice of treatment for patients with type 2 diabetes (T2D). However, there is a need for more effective tools and technologies to facilitate structured lifestyle interventions and to ensure a better compliance, sustainability, and health benefits of exercise training in patients with T2D. The InterWalk initiative and its innovative application (app) for smartphones described in this study were developed by the Danish Centre for Strategic Research in T2D aiming at implementing, testing, and validating interval walking in patients with T2D in Denmark. The interval walking training approach consists of repetitive 3-minute cycles of slow and fast walking with simultaneous intensity guiding, based on the exercise capacity of the user. The individual intensity during slow and fast walking is determined by a short initial self-conducted and audio-guided fitness test, which combined with automated audio instructions strives to motivate the individual to adjust the intensity to the predetermined individualized walking intensities. The InterWalk app data are collected prospectively from all users and will be linked to the unique Danish nationwide databases and administrative registries, allowing extensive epidemiological studies of exercise in patients with T2D, such as the level of adherence to InterWalk training and long-term effectiveness surveys of important health outcomes, including cardiovascular morbidity and mortality. Currently, the InterWalk app has been downloaded by >30,000 persons, and the achieved epidemiological data quality is encouraging. Of the 9,466 persons providing personal information, 80% of the men and 62% women were overweight or obese (body mass index ≥25). The InterWalk project represents a contemporary technology-driven public health approach to monitor real-life exercise adherence and to propagate improved health through exercise intervention in T2D and in the general population. Keywords: exercise, telemedicine, cell phones

Immunotherapy of colorectal carcinoma (CRC) has great promise as the presence of T lymphocytes in CRC tissues in situ is correlated with reduced recurrence and increased survival. Thus, identification of the antigens recognized by T cells of CRC patients may permit development of vaccines with potential benefit for these patients. Using expression cloning, we identified the antigen, nucleophosmin (Npm), recognized by an HLA-A1 restricted cytotoxic T lymphocyte (CTL) line derived from the peripheral blood mononuclear cells (PBMC) of a rectal cancer patient. A decamer peptide derived from the Npm sequence sensitized peptide-pulsed HLA-A1 positive cells to lysis by the CTL line. The peptide also induced proliferative and cytotoxic T lymphocytes in the PBMC of 4 of 6 CRC patients, which lysed HLA-A1 positive peptide-pulsed target cells and CRC cells endogenously expressing Npm. Overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.

Background— Coronary drug-eluting stents with biodegradable polymers have been designed to improve safety and efficacy. Methods and Results— The Scandinavian Organization for Randomized Trials With Clinical Outcome (SORT OUT) VII trial—a large-scale registry-based randomized, multicenter, single-blind, 2-arm, noninferiority trial—compared 2 biodegradable polymer drug-eluting stents: the thin-strut cobalt–chromium sirolimus-eluting Orsiro stent and the stainless steel biolimus-eluting Nobori stent in an all-comer patient population. The primary end point target lesion failure was a composite of cardiac death, myocardial infarction (not related to other than index lesion), or target lesion revascularization within 1 year, analyzed by intention to treat (noninferiority margin of 3.0%). Clinically driven event detection based on Danish registries was used. A total of 1261 patients were assigned to receive the sirolimus-eluting stent (1590 lesions) and 1264 patients to the biolimus-eluting stent (1588 lesions). At 1 year, the composite end point target lesion failure occurred in 48 patients (3.8%) in the sirolimus-eluting group and in 58 patients (4.6%) in the biolimus-eluting group (absolute risk difference, −0.78% [upper limit of 1-sided 95% confidence interval, 0.61%]; P P =0.034), which largely was attributable to a lower risk of subacute definite stent thrombosis: 0.1% versus 0.6% (rate ratio, 0.12; 95% confidence interval, 0.02–1.00; P =0.05). Conclusions— The thin-strut sirolimus-eluting Orsiro stent was noninferior to the biolimus-eluting Nobori stent in unselected patients for target lesion failure at 1 year. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT01879358.

BACKGROUND: The excess risk of antibiotic use and hospital-treated infections in patients with type 2 diabetes (T2D) compared with general population is poorly understood.METHODS: In a nationwide cohort of patients with incident T2D (n = 155 158) and an age-, gender-, and residence-matched comparison cohort (n = 774 017), we used Cox regression to compute rates and confounder-adjusted rate ratios (aRRs) of community-based antibiotic prescription redemption and hospital-treated infections during 2004-2012.RESULTS: The rates of community-based antibiotic prescriptions in the T2D and comparison cohorts were 364 vs 275 per 1000 person-years after a median follow-up of 1.1 years (aRR = 1.24; 95% confidence interval [CI], 1.23 to 1.25). The corresponding rates for hospital-treated infection were 58 vs 39 per 1000 person-years after a median follow-up of 2.8 years (aRR = 1.49; 95% CI, 1.47 to 1.52). The aRRs were increased particularly for urinary tract infections (UTIs, 1.41; 95% CI, 1.35 to 1.45), skin infections (1.50; 95% CI, 1.45 to 1.55), septicemia (1.60; 95% CI, 1.53 to 1.67), and tuberculosis (1.61; 95% CI, 1.25 to 2.06) and of community-based antibiotics prescribed for UTIs (1.31; 95% CI, 1.29 to 1.33), Staphylococcus aureus infections (1.32; 95% CI, 1.30 to 1.34), and mycobacterial infections (1.69; 95% CI, 1.36 to 2.09). The 1-year aRR declined from 1.89 (95% CI, 1.75 to 2.04) in 2004 to 1.59 (95% CI, 1.45 to 1.74) in 2011 for hospital-treated infection (trend P = .007) and from 1.31 (95% CI, 1.27 to 1.36) in 2004 to 1.26 (95% CI, 1.22 to 1.30) in 2011 for community-based antibiotic prescriptions (trend P = .006).CONCLUSIONS: Patients with T2D have rates of community-based antibiotic prescriptions and hospital-treated infections that are higher than for the general population.

Long-term safety and efficacy for everolimus-eluting stents (EES) versus those of sirolimus-eluting stents (SES) are unknown.This study compared 5-year outcomes for EES with those for SES from the SORT OUT IV (Scandinavian Organization for Randomized Trials with Clinical Outcome) trial.Five-year follow-up was completed for 2,771 patients (99.9%). Primary endpoint was a composite of major adverse cardiac events (MACE), including cardiac death, myocardial infarction (MI), target vessel revascularization (TVR), and definite stent thrombosis.At 5-years, MACE occurred in 14.0% and 17.4% in the EES and SES groups, respectively (hazard ratio [HR]: 0.80, 95% confidence interval [CI]: 0.66 to 0.97; p = 0.02). The MACE rate did not differ significantly within the first year (HR: 0.96, 95% CI: 0.71 to 1.19; p = 0.79), but from years 1 through 5, the MACE rate was lower with EES (HR: 0.71, 95% CI: 0.55 to 0.90; p = 0.006; p interaction = 0.12). Definite stent thrombosis was lower with EES (0.4%) than with SES (2.0%; HR: 0.18, 95% CI: 0.07 to 0.46), with a lower risk of very late definite stent thrombosis in the EES group (0.2% vs. 1.4%, respectively; HR: 0.16, 95% CI: 0.05 to 0.53). When censoring the patients at the time of stent thrombosis, we found no significant differences between the 2 stent groups for MACE rates (HR: 0.89, 95% CI: 0.73 to 1.08; p = 0.23), target lesion revascularization (HR: 0.90, 95% CI: 0.64 to 1.27; p = 0.55), and MI (HR: 0.93, 95% CI: 0.64 to 1.36; p = 0.72).At 5-year follow-up, MACE rate was significantly lower with EES- than with SES-treated patients, due largely due to a lower risk of very late definite stent thrombosis. (Randomized Clinical Comparison of the Xience V and the Cypher Coronary Stents in Non-selected Patients With Coronary Heart Disease [SORT OUT IV]; NCT00552877).

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.

Development of a vaccine for prevention of congenital cytomegalovirus (CMV) disease is a priority. This study evaluated a "prime-boost" strategy by comparing the safety and immunogenicity of 3 doses of subunit CMV glycoprotein B (gB) vaccine plus MF59 (a squalene-in-water emulsion), 2 doses of a canarypox recombinant vaccine expressing CMVgB (ALVAC-CMVgB) followed by 2 doses of the subunit gB vaccine, 3 doses of both vaccines administered concomitantly, and placebo in 105 healthy, CMV-seronegative adults. Systemic adverse events were rare, but local reactions were common in all groups. After the first subunit vaccination, neutralizing antibody titers in the prime-boost group were comparable to those in subjects receiving 2 subunit vaccinations, indicating a priming effect of ALVAC-CMVgB. However, after the final dose, antibody and cell-mediated immune responses were not significantly different among the groups. All 3 vaccine regimens induced high-titer antibody and lymphoproliferative responses, but no benefit for priming or simultaneous vaccination was detected.

The prevalence of human cytomegalovirus (HCMV) pp65-, pp150-, IE1-exon4-, gB- and pp28-specific cytotoxic T lymphocyte (CTL) responses was compared among 34 healthy individuals, grouped by neutralizing antibody titers. Moderately and highly seropositive donors showed predominantly pp65- and IE1-exon4-specific CTL responses (92% and 76% of the donors, respectively), with similar precursor frequencies in the 2 donors tested. In addition, highly seropositive and a few moderately seropositive donors showed CTL responses to gB and pp150 (33% and 30% of the donors, respectively). No individual recognized pp28 as a target in the CTL assay. Phenotypic analysis revealed a mixed effector population of CD4+ and CD8+ (1 donor) or only CD8+ cells for pp65-specific effectors (2 donors). IE1-exon4- and pp150-specific effectors were CD8+ (2 donors and 1 donor, respectively), whereas gB-specific CTLs were CD4+ (1 donor). These data may help to design a cellular immunity-based vaccine effective against HCMV diseases.

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.

Anil Mor, Sinna P Ulrichsen, Elisabeth Svensson, Klara Berencsi, Reimar W Thomsen Department of Clinical Epidemiology, Institute of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark Background: To reduce the increasing burden of pneumonia hospitalizations, we need to understand their determinants. Being married may decrease the risk of severe infections, due to better social support and healthier lifestyle. Patients and methods: In this population-based case-control study, we identified all adult patients with a first-time pneumonia-related hospitalization between 1994 and 2008 in Northern Denmark. For each case, ten sex- and age-matched population controls were selected from Denmark's Civil Registration System. We performed conditional logistic regression analysis to estimate the odds ratios (ORs) for pneumonia hospitalization among persons who were divorced, widowed, or never married, as compared with married persons, adjusting for age, sex, 19 different comorbidities, alcoholism-related conditions, immunosuppressant use, urbanization, and living with small children. Results: The study included 67,162 patients with a pneumonia-related hospitalization and 671,620 matched population controls. Compared with controls, the pneumonia patients were more likely to be divorced (10% versus 7%) or never married (13% versus 11%). Divorced and never-married patients were much more likely to have previous diagnoses of alcoholism-related conditions (18% and 11%, respectively) compared with married (3%) and widowed (6%) patients. The adjusted OR for pneumonia-related hospitalization was increased, at 1.29 (95% confidence interval [CI]: 1.25-1.33) among divorced; 1.15 (95% CI: 1.12-1.17) among widowed; and 1.33 (95% CI: 1.29-1.37) among never-married individuals as compared with those who were married. Conclusion: Married individuals have a decreased risk of being hospitalized with pneumonia compared with never-married, divorced, and widowed patients. Keywords: marital status, risk, mortality, immune function

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.

Background Infiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells. Methods To identify chemokine and chemokine receptors involved in T-cell migration toward CRC cells, we have used our previously published three-dimensional organotypic CRC culture system. Organotypic culture was initiated with a layer of fetal fibroblast cells mixed with collagen matrix in a 24 well tissue culture plate. A layer of CRC cells was placed on top of the fibroblast-collagen layer which was followed by a separating layer of fibroblasts in collagen matrix. Anti-CRC specific cytotoxic T lymphocytes (CTLs) mixed with fibroblasts in collagen matrix were placed on top of the separating layer. Excess chemokine ligand (CCL) or Abs to chemokine or chemokine receptor (CCR) were used in migration inhibition assays to identify the chemokine and the receptor involved in CTL migration. Results Inclusion of excess CCL2 in T-cell layer or Ab to CCL2 in separating layer of collagen fibroblasts blocked the migration of CTLs toward tumor cells and in turn significantly inhibited tumor cell apoptosis. Also, Ab to CCR2 in the separating layer of collagen and fibroblasts blocked the migration of CTLs toward tumor cells and subsequently inhibited tumor cell apoptosis. Expression of CCR2 in four additional CRC patients' lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients. Conclusions Our data suggest that CCL2 secreted by tumor cells and CCR2 receptors on CTLs are involved in migration of CTLs towards tumor. Gene therapy of tumor cells with CCL2 or CCL2/anti-tumor Ab fusion proteins may attract CTLs that potentially could inhibit tumor growth.

A biotechnological approach to the immunomodulating ability of various phenothiazines and related tricyclic compounds in vitro and in vivo was made. The effect of these compounds was studied on various mononuclear leukocyte functions such as antibody-dependent cell-mediated cytotoxicity, blast transformation of lymphocytes by three mitogens, phytohemagglutinin, concanavalin A and pokeweed mitogen (PWM), rosette formation with erythrocytes and with antibody coated erythrocytes (EA), as well as on polymorphonuclear leukocyte functions including phagocytosis and chemiluminescence emission. Moreover, the ability of these compounds to modulate the bioluminescence emission capacity of Escherichia coli cloned with lux genes was measured. Also in vivo studies were made by measuring the number of haemolytic plaque forming cells in the spleen of mice after drug administration. The electrochemical structure of these tricyclic compounds was shown to be in good correlation with their immunomodulating activity on all the functions except EA rosette formation and on blast transformation by PWM showing the role of charge transfer complex formation in the drug-target cell interaction. These results suggest that the biological effectiveness of phenothiazines and related tricyclic compounds can be presumed from their electrochemical properties and a tricyclic compound with a given biological effect can be theoretically designed.

Our previous analysis of the role of chemokines in T lymphocyte trafficking toward human tumor cells revealed the migration of a melanoma patient's cytotoxic T lymphocytes (CTL) toward autologous tumor cells, resulting in tumor cell apoptosis, in an organotypic melanoma culture. CTL migration was mediated by CX chemokine receptor (CXCR) 4 expressed by the CTL and CX chemokine ligand (CXCL) 12 secreted by the tumor cells, as evidenced by blockage of CTL migration by antibodies to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. Here, we present the results of T cell migration in one additional melanoma patient and T cell and tumor cell analyses for CXCR4 and CXCL12 expression, respectively, in 12 additional melanoma patients, indicating the preferential role of CXCR4 and CXCL12 in CTL migration toward melanoma cells. These studies add to the increasing body of evidence suggesting that CXCL12 is a potent chemoattractant for T cells.

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient’s CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1α) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.

The effect of infection with Chlamydia pneumoniae on host messenger RNA expression in human monocytic cells with complement DNA microarrays was studied. The data chronicle a cascade of transcriptional events affecting 128 genes, many of which have not previously been reported to be affected by C. pneumoniae infection. Down-regulated genes are primarily associated with RNA and DNA metabolism, chromosomal stability, and cell-cycle regulation. Up-regulated messages include those for a variety of genes with important proinflammatory functions. Many of the up-regulated genes-including the hyaluron receptor CD44, vasoconstrictor endothelin-1, smooth muscle growth factor heparin-binding EGF-like growth factor, and fatty acid binding protein-4-had been previously described as linked to the development of atherosclerosis and other chronic inflammatory diseases. C. pneumoniae-infected monocytes can contribute to the development and progression of diseases for which acute or chronic inflammation has been shown to be important, such as atherosclerosis.

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

BACKGROUND: We aimed to examine the prevalence of and modifiable factors associated with elevated C-reactive Protein (CRP), a marker of inflammation, in men and women with newly diagnosed Type 2 Diabetes mellitus (DM) in a population-based setting.METHODS: CRP was measured in 1,037 patients (57% male) with newly diagnosed Type 2 DM included in the prospective nationwide Danish Centre for Strategic Research in Type 2 Diabetes (DD2) project. We assessed the prevalence of elevated CRP and calculated relative risks (RR) examining the association of CRP with lifestyle and clinical factors by Poisson regression, stratified by gender. We used linear regression to examine the association of CRP with other biomarkers.RESULTS: The median CRP value was 2.1 mg/L (interquartile range, 1.0 - 4.8 mg/L). In total, 405 out of the 1,037 Type 2 DM patients (40%) had elevated CRP levels (>3.0 mg/L). More women (46%) than men (34%) had elevated CRP. Among women, a lower risk of elevated CRP was observed in patients receiving statins (adjusted RR (aRR) 0.7 (95% confidence interval (CI) 0.6-0.9)), whereas a higher risk was seen in patients with central obesity (aRR 2.3 (95% CI 1.0-5.3)). For men, CRP was primarily elevated among patients with no regular physical activity (aRR 1.5 (95% CI 1.1-1.9)), previous cardiovascular disease (aRR1.5 (95% CI 1.2-1.9) and other comorbidity. For both genders, elevated CRP was 1.4-fold increased in those with weight gain >30 kg since age 20 years. Sensitivity analyses showed consistent results with the full analysis. The linear regression analysis conveyed an association between high CRP and increased fasting blood glucose.CONCLUSIONS: Among newly diagnosed Type 2 DM patients, 40% had elevated CRP levels. Important modifiable risk factors for elevated CRP may vary by gender, and include low physical activity for men and central obesity and absence of statin use for women.

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with Chlamydia pneumoniae .

Background —Studies have suggested that the prevalence of antibodies against heat-shock proteins (HSPs), Chlamydia pneumoniae ( Cpn ), and cytomegalovirus (CMV) is associated with coronary artery disease (CAD), but the independent or joint effects of human (h) HSP60 antibodies and these pathogens in patients have not been fully elucidated. Methods and Results —A total of 405 subjects (276 patients with CAD and 129 control individuals) were tested for serum antibodies to hHSP60, Cpn , and CMV immediate-early-1 (IE1) antigens. Patients were also assessed for serum cholesterol, triglyceride levels, and smoking habit. Significantly elevated levels of antibodies to hHSP60 and Cpn but not to CMV-IE1 antigens were documented in CAD patients. Multiple logistic regression analysis and subanalyses of selected subjects showed that these associations were independent of age, sex, smoking, and serum lipid levels. Antibodies to hHSP60 and Cpn did not correlate quantitatively; however, the relative risk of disease development was substantially increased in subjects with high antibody levels to both hHSP60 and Cpn , reaching an odds ratio of 82.0 (95% CI 10.6 to 625.0). Conclusions —High levels of antibodies to hHSP60 and Cpn are independent risk factors for coronary atherosclerosis, but their simultaneous presence substantially increases the risk for disease development.

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8 + . Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.

Chlamydia pneumoniae is a common cause of pulmonary infection, with serum positivity in at least 50% of the general population. In this study, we report that human PBMCs exposed to C. pneumoniae are resistant to apoptosis induced by the potent photoactivated chemotherapeutic agents 8-methoxypsoralen and hypericin. In contrast, PBMCs treated with a heat-inactivated inoculum exhibit normal susceptibility to apoptosis. We also observed that human PBMCs are responsive to C. pneumoniae infection by secretion of key immune regulatory cytokines, including IL-12 and IL-10. While IL-12 may play an important role in limiting C. pneumoniae proliferation within cells, IL-10 serves an anti-inflammatory function by down-regulating proinflammatory cytokines such as IL-12 and TNF-α. Depletion of endogenous IL-10, but not of IL-12, abolished the apoptosis resistance of C. pneumoniae-infected PBMCs. Furthermore, addition of exogenous IL-10, but not IL-12, significantly increased the resistance of control inoculum-treated PBMCs to photoactivated 8-methoxypsoralen- and hypericin-induced apoptosis. Therefore, we conclude that C. pneumoniae possesses an antiapoptotic mechanism. The resistance to apoptosis observed in PBMCs exposed to C. pneumoniae is due, at least partially, to the IL-10 induced during C. pneumoniae infection.

BALB/c and strain 129 mice infected intranasally withChlamydia pneumoniaedisplayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-γ production and in vitro spleen cell IFN-γ production in response to eitherC. pneumoniaeor concanavalin A. In γ-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. FollowingC. pneumoniaeinfection, 129 mice lacking the IFN-γ receptor α chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, duringC. pneumoniaelung infection, IL-12, more than IFN-γ, plays a role in pulmonary-cell infiltration. IFN-γ and IL-12, acting mostly through its induction of IFN-γ and Th1 responses, play an important role in controlling acuteC. pneumoniaeinfection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-γ-independent mechanisms.

A murine model of the cytotoxic T lymphocyte (CTL) response to glycoprotein B (gB) of human cytomegalovirus (HCMV) was developed based on the use of adenovirus (Ad) and vaccinia virus (Vac) recombinants expressing gB. Mice of different major histocompatibility haplotypes [CBA (H-2k), BALB/k (H-2k) and BALB/c (H-2d)] infected with the Ad-gB recombinant developed an Ad-specific CTL response. However, only the H-2k mice developed a significant HCMV gB-specific CTL response, as indicated by the major histocompatibility complex class I-restricted lysis of Vac strain Copenhagen (VacC)-gB recombinant-infected target cells by H-2k mouse immune spleen cells. The VacC-gB recombinant elicited only a weak gB-specific CTL response in these mice, indicating that the observed gB-specific CTL response in mice is dependent on the expression vector used for immunization. The gB-specific cytotoxicity observed in H-2k mice was mediated by the CD8 lymphocyte subset.

In light of the important role of apoptotic cell death in the pathogenesis of several viral infections, we asked whether the cytopathogenicity evoked by rubella virus (RV) might also involve apoptotic mechanisms. The To-336 strain of RV induced apoptosis in Vero and RK-13 cells, but not in fibroblast cell lines. UV-inactivated RV virions did not elicit the apoptotic response, indicating that productive infection is required for the induction of cell death. Both p53 and p21 protein levels were highly elevated in RV-infected Vero cells. The level of p21 mRNA was increased, while expression of the p53 gene was unaffected by RV infection. A dominant-negative p53 mutant (p53 W248 ) conferred partial protection from RV-induced apoptosis. These data implicate a p53-dependent apoptotic pathway in the cytopathogenicity of RV, thereby suggesting a mechanism by which RV exerts its teratogenic effects.