Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay.

Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naïve 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the beta-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PCA is a promising methodology for stringent selection of binders from complex naïve libraries to yield high affinity reagents with selective target binding characteristics.

Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.

Rosiglitazone and carotid IMT progression rate in a mixed cohort of patients with type 2 diabetes and the insulin resistance syndrome: main results from the Rosiglitazone Atherosclerosis Study.

OBJECTIVE: Insulin resistance is associated with progression of atherosclerosis. We assessed the effect of 12 months of treatment with rosiglitazone (RSG) on the progression of carotid intima-media thickness (IMT) in people with type 2 diabetes mellitus (T2DM) or the insulin resistance syndrome (IRS). DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Malmö University Hospital, Malmö, Sweden. SUBJECTS: 555 subjects (200 with T2DM and 355 nondiabetics with IRS according to EGIR criteria), aged 35-80 years. 447 subjects (165 T2DM and 282 IRS) completed the study. INTERVENTION: Participants were allocated to placebo or RSG 4 mg for 2 months and then 8 mg daily. MAIN OUTCOME MEASURE: Change in composite IMT [mean IMT in the common carotid artery (CCA) and maximal IMT in the bulb] was the primary and various other IMT measures were secondary outcome variables. RESULTS: There was no effect of RSG treatment in the mixed population. In T2DM patients there was a reduced progression of the composite IMT (mean change: 0.041 vs. 0.070 mm, P = 0.07), and of the mean IMT CCA (mean change: -0.005 mm vs. 0.021 mm, P = 0.007). RSG treatment led to significant reductions of HOMA-IR, fasting plasma glucose, HbA1c, PAI-1 activity, fibrinogen, C-reactive protein and matrix metalloproteinase-9. CONCLUSIONS: In a mixed study population of patients with T2DM and IRS RSG treatment was not associated with a statistically significant reduction of carotid IMT progression rate. Separate analyses of these two patient groups indicated, however, a significant beneficial effect on CCA IMT in T2DM patients but no similar effect in subjects with IRS.

Area social characteristics and carotid atherosclerosis.

OBJECTIVES: To explore the effect of social characteristics of residential areas on carotid atherosclerosis prevalence. METHODS AND RESULTS: The associations among area social characteristics and B-mode ultrasound determined carotid plaque-score (a semi-quantitative scale measuring the degree of atherosclerosis in the carotid bifurcation area) were cross-sectionally investigated in a general population sample of 4033 men and women. Area socioeconomic circumstances were described through a social deprivation index calculated from migration rate, percentage residents with foreign citizenship among those with foreign background, dependency on social welfare support, and employment rate. Living in socially deprived areas was associated with an increased carotid plaque-score in both men (P for trend = 0.004) and women (P for trend = 0.007). These associations were only slightly reduced after adjustment for individual level indicators with a decrease of the absolute mean difference in carotid plaque-score between worse-off and better-off areas of 9% for men and 13% for women, whereas adjustment for risk factors turned the trend non-significant in women, however, not in men. CONCLUSIONS: Those living in socially deprived areas in general had more extensive carotid atherosclerosis. However, in these areas there were a substantial number of individuals with low degrees of carotid atherosclerosis and vice versa. Thus, with regard to conceptual ideas of causal inference, the social characteristics of an area seem to be associated with the prevalence of carotid atherosclerosis. However, with regard to benefits of prevention, focusing on geographical areas would probably give a restricted benefit, where only some high-risk individuals would be reached.

Multi-factor dimensionality reduction applied to a large prospective investigation on gene-gene and gene-environment interactions.

It is becoming increasingly evident that single-locus effects cannot explain complex multifactorial human diseases like cancer. We applied the multi-factor dimensionality reduction (MDR) method to a large cohort study on gene-environment and gene-gene interactions. The study (case-control nested in the EPIC cohort) was established to investigate molecular changes and genetic susceptibility in relation to air pollution and environmental tobacco smoke (ETS) in non-smokers. We have analyzed 757 controls and 409 cases with bladder cancer (n=124), lung cancer (n=116) and myeloid leukemia (n=169). Thirty-six gene variants (DNA repair and metabolic genes) and three environmental exposure variables (measures of air pollution and ETS at home and at work) were analyzed. Interactions were assessed by prediction error percentage and cross-validation consistency (CVC) frequency. For lung cancer, the best model was given by a significant gene-environment association between the base excision repair (BER) XRCC1-Arg399Gln polymorphism, the double-strand break repair (DSBR) BRCA2-Asn372His polymorphism and the exposure variable 'distance from heavy traffic road', an indirect and robust indicator of air pollution (mean prediction error of 26%, P<0.001, mean CVC of 6.60, P=0.02). For bladder cancer, we found a significant 4-loci association between the BER APE1-Asp148Glu polymorphism, the DSBR RAD52-3'-untranslated region (3'-UTR) polymorphism and the metabolic gene polymorphisms COMT-Val158Met and MTHFR-677C>T (mean prediction error of 22%, P<0.001, mean CVC consistency of 7.40, P<0.037). For leukemia, a 3-loci model including RAD52-2259C>T, MnSOD-Ala9Val and CYP1A1-Ile462Val had a minimum prediction error of 31% (P<0.001) and a maximum CVC of 4.40 (P=0.086). The MDR method seems promising, because it provides a limited number of statistically stable interactions; however, the biological interpretation remains to be understood.

The role of low grade inflammation as measured by C-reactive protein levels in the explanation of socioeconomic differences in carotid atherosclerosis.

BACKGROUND: The role of inflammation as part of the explanation of socioeconomic differences in carotid atherosclerosis has not been specifically investigated. METHODS AND RESULTS: The associations between socioeconomic position (SEP), C-reactive protein (CRP), and preclinical carotid atherosclerosis were investigated in a general population sample of 3921 middle-aged Swedish men and women. Common carotid intima-media thickness (IMT) and presence of carotid plaque (focal IMT > 1.2 mm) were determined by B-mode ultrasound. The results showed that low SEP was associated with increased levels of CRP, independently of established risk factors. Furthermore, common carotid IMT increased with increasing CRP-levels. Presence of carotid plaque increased with increasing CRP-levels in men, but not in women. While the socioeconomic differences in carotid IMT were weak, there were associations between low educational level and carotid plaque prevalence with an age- and sex-adjusted odds ratio (OR) of 1.39 (95% CI: 1.21, 1.59). A similar association was seen for having a manual occupation, OR = 1.23 (95% CI: 1.07, 1.42). The age- and sex-adjusted absolute differences in carotid plaque prevalence were 9% with regard to educational level and 7% with regard to occupational status. Adjustment for CRP caused only a minor attenuation of the association between SEP and carotid atherosclerosis. CONCLUSIONS: The association between SEP and carotid atherosclerosis as measured by carotid IMT and carotid plaque could only to a minor extent be referred to differences in low grade inflammation as measured by CRP.

High incidence of vascular reconstructions in socioeconomically deprived areas of an urban Swedish population.

BACKGROUND: In Malmö approximately 250,000 citizens live in 17 administrative areas with substantial socioeconomic differences. At the single centre for arterial reconstruction, Malmö University Hospital, all procedures are registered prospectively. METHODS: Between 1987 and 2002, 1832 Malmö citizens underwent reconstruction for peripheral arterial disease, either intermittent claudication (IC) or critical leg ischaemia (CLI). A socioeconomic score based on migration rate, percentage of residents with foreign citizenship/residents with foreign background, social welfare support dependency and unemployment rate has previously been developed and validated for each area. The relationship between socioeconomic score and area-specific standardized morbidity ratios (SMRs) after vascular reconstruction was analysed by population-weighted linear regression. RESULTS: The mean incidence of vascular reconstruction was 76 (range 27-106) per 100,000 person years. Age- and sex-adjusted SMRs ranged from 0.57 to 1.39. A strong correlation between SMR and socioeconomic score was found overall (R=0.63; P=0.007), in men (R=0.63; P=0.007) and in women (R=0.58; P=0.039), and for IC (R=0.58; P=0.015) and CLI (R=0.58; P=0.015). CONCLUSION: In an urban population with similar access to medical care, vascular reconstruction rates varied substantially. High-rate areas were characterized by inferior socioeconomic circumstances and a higher prevalence of smoking, hypertension and obesity.

Plasma levels of complement C3 is associated with development of hypertension: a longitudinal cohort study.

Hypertension has been associated with raised plasma levels of complement factor 3 and 4 (C3 and C4). The nature of this association is unclear. This population-based longitudinal study explored whether C3 or C4 is associated with development of hypertension. Blood pressure and plasma levels of C3 and C4 were determined in 2178 healthy men, aged 35-50 years, initially without treatment for hypertension. Incidence of hypertension and blood pressure increase over 15.7 (+/-2.2) years follow-up was studied in relation to C3 and C4 at baseline. Among men with initially normal blood pressure (<160/95 mm Hg), incidence of hypertension (>or=160/95 mm Hg or treatment) was 32, 42, 37 and 47%, respectively, for men with C3 in the first, second, third and fourth quartile (trend: P=0.001). This relationship remained significant after adjustment for confounding factors. Among men without blood pressure treatment, systolic BP increase (mean+standard error, adjusted for age, initial blood pressure and follow-up time) was 17.5+0.8, 19.6+0.9, 19.8+0.8 and 20.8+0.8 mm Hg, respectively, in the C3 quartiles (trend: P=0.004). C3 was not associated diastolic blood pressure at follow-up. Although C4 was associated with blood pressure at the baseline examination, there was no relationship between C4 and development of hypertension or future blood pressure increase. It is concluded that C3 in plasma is associated with future blood pressure increase and development of hypertension.

Reduced lung function predicts increased fatality in future cardiac events. A population-based study.

OBJECTIVE: Moderately reduced lung function in apparently healthy subjects has been associated with incidence of coronary events. However, whether lung function is related to the fatality of the future events is unknown. This study explored whether reduced forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV(1)) in initially healthy men is related to the fatality of the future coronary events. DESIGN: Prospective cohort study. SETTING: Population-based study from Malmö, Sweden. SUBJECTS: A total of 5452 healthy men, 28-61 years of age. MAIN OUTCOME MEASURES: Incidence of first coronary events was monitored over a mean follow-up of 19 years. The fatality of the future events was studied in relation to FEV and FVC. RESULTS: A total of 589 men suffered a coronary event during follow-up, 165 of them were fatal during the first day. After risk factors adjustment, low FEV or FVC were associated with incidence of coronary events (fatal or nonfatal) and this relationship was most pronounced for the fatal events. Amongst men who subsequently had a coronary event, the case-fatality rates were higher in men with low FEV or FVC. Adjusted for risk factors, the odds ratio for death during the first day was 1.00 (reference), 1.63 (95% CI: 0.9-3.1), 1.86 (1.0-3.5) and 2.06 (1.1-3.9), respectively, for men with FVC in the 4th, 3rd, 2nd, and lowest quartiles (trend: P < 0.05). FEV showed similar relationships with the fatality rates. CONCLUSION: Apparently healthy men with moderately reduced lung function have higher fatality in future coronary events, with a higher proportion of coronary heart disease deaths and less nonfatal myocardial infarction.

Fatality of acute coronary events in relation to hypertension and low-grade inflammation: a population-based cohort study.

Hypertension has been associated with increased case-fatality rates among individuals who subsequently suffer from acute coronary events. It is unknown whether inflammation modifies this relationship. This population-based study explored the effects of inflammation and hypertension on incidence of coronary event, and on the fatality of the future events. Blood pressure (BP) and five inflammation-sensitive plasma proteins (ISPs, fibrinogen, orosomucoid, alpha 1-antitrypsin, haptoglobin and ceruloplasmin) were determined in 6071 healthy men. During the mean follow-up of 19 years, 679 men had a first coronary event (non-fatal myocardial infarction or death from coronary heart disease). Of them, 197 (29%) were fatal cases (death during the first day). As expected, hypertension was associated with increased incidence of coronary events and increased proportion of fatal cases. At all levels of BP, high ISPs (> or =2 ISPs in top quartile) significantly added to the incidence of events. Men with high ISPs had the highest case-fatality rates. The difference in case-fatality rate between men with and without high ISPs was, however, significant only in men with normal BP (<130/85 mm Hg) (33 vs 19%, P < 0.05), and not in men with moderate or severe hypertension (> or =160/100 mm Hg) (40 vs 35%, P = 0.32). High ISPs add to the incidence of coronary events at all levels of BP. Hypertension and inflammation are both independently associated with increased case-fatality in subjects who later have an acute coronary event. The influence of ISPs on the case-fatality rate seems to be most important in men with normal BP.

Sex differences in the relationships between BMI, WHR and incidence of cardiovascular disease: a population-based cohort study.

OBJECTIVE: Body mass index (BMI) is associated with increased incidence of cardiovascular disease (CVD). However, the risk could be very different for individuals with the same body mass. The present study explored whether regional fat distribution, as measured by waist-hip ratio (WHR), could modify the impact of BMI on the risk of CVD in men and women. DESIGN: Prospective population-based study. SUBJECTS: A total of 10 369 men and 16 638 women, 45-73 years old, from general population in Malmö, Sweden. MEASUREMENTS: All subjects were followed over 7 years for the incidences of first-ever cardiac event (CE) and ischemic stroke in relation to BMI category (<25.0, 25.0-29.9, > or =30.0) and WHR. RESULTS: The prevalence of overweight and obesity was 39.4 and 13.0%, respectively. During follow-up, 1280 subjects suffered a CVD event (750 CE, 530 ischemic stroke). The risk of CVD in women increased with increasing levels of WHR, irrespective of BMI category. In men, WHR (per 1 s.d. increase) was associated with increased incidence of CVD in those with normal weight (relative risk (RR)=1.24; 95% CI: 1.13-1.37) after adjustments for confounding factors. However, WHR was not related to CVD in overweight men (RR=1.06; 95%CI: 0.94-1.20) or obese men (RR=1.04; 95%CI: 0.87-1.24). A significant interaction was observed between sex and WHR on the CVD risk. CONCLUSION: The effect of WHR on incidence of CVD is modified by the overall body weight and by gender. WHR adds prognostic information on the cardiovascular risk in women at all levels of BMI, and in men with normal weight.

Survival rate 28 days after hospital admission with first myocardial infarction. Inverse relationship with socio-economic circumstances.

OBJECTIVE: To study to what extent geographical differences of the mortality from ischaemic heart disease (IHD) can be accounted for by the 28-day case fatality rate (CFR) following first hospital admittance for acute myocardial infarction (MI) and whether the geographical pattern of survival has any relationship with socio-economic circumstances. DESIGN: Register-based surveillance study. SETTING: Seventeen residential areas in Malmö, Sweden. SUBJECTS: All 5533 patients were admitted during 1986-1995 for a first acute MI at Malmö University Hospital. Main outcome measures. CFR is based on record linkage with national registers. Area-specific cardiovascular and socio-economic scores (SES) are based on previous cross-sectional studies. RESULTS: In patients below 75 years of age, differences of the 28-day CFR accounted for 20-30% of the geographical variance in mortality from IHD. No corresponding association was found in older age groups. Patients from areas with low SES had the highest CFR, 23.8%. The odds ratios of fatal outcome for patients from areas with median and low SES (versus high SES) were 1.23 (95% CI: 1.01-1.50) and 1.25 (95% CI: 1.03-1.52), respectively (P for trend: 0.060). The strongest correlation was observed in men below 75 years of age (P for trend: 0.007). During the study period there was an improvement of the survival rate for patients from high and medium SES areas but no corresponding change for patients coming from areas having a low SES. CONCLUSIONS: In patients below 75 years, geographical differences of the mortality from IHD were related to differences of the 28-day CFR following hospital admittance for a first MI. Rates of survival were inversely related to socio-economic circumstances in the patient's residential area.

Weight gain in relation to plasma levels of complement factor 3: results from a population-based cohort study.

AIMS/HYPOTHESIS: Mice that are deficient for complement factor 3 (C3) have shown resistance to weight gain, despite increased food intake. Cross-sectional studies of humans have reported correlations between C3 and obesity. This longitudinal study explored whether C3 predicts a large weight gain in middle-aged men. METHODS: Plasma concentrations of C3 and complement factor 4 (C4) were measured in 2,706 non-diabetic healthy men aged between 38 and 50 years, who were re-examined after a mean period of 6.1 years. RESULTS: After adjustments for initial weight, age, height and follow-up time, the odds of incurring large weight gain (75th percentile, > or =3.8 kg) were 1.00 (reference), 0.96 (95% CI:0.7-1.2), 1.1 (CI:0.9-1.5) and 1.4 (CI:1.1-1.8), respectively, among men with C3 levels in the first, second, third and fourth quartiles (p for trend=0.01) respectively. This relationship remained significant after further adjustments for lifestyle factors (physical inactivity, alcohol, smoking), metabolic factors (glucose or homeostasis model assessment values, cholesterol, triglycerides), inflammatory markers (fibrinogen, haptoglobin, ceruloplasmin, orosomucoid, alpha1-antitrypsin) and for C4. C4 was associated with weight gain after adjustments for initial weight, height, follow-up time and lifestyle factors, but not after adjustments for C3. CONCLUSIONS/INTERPRETATION: C3 is a risk factor for incurring large weight gain in middle-aged men.

Incident coronary events and case fatality in relation to common carotid intima-media thickness.

OBJECTIVES: To evaluate the incidence of coronary events (CE) and case fatality in relation to common carotid intima-media thickness (IMT) and carotid plaque over a median follow up of 7 years. SUBJECTS: A total of 5163 Swedish middle-aged men and women with no prior myocardial infarction and/or stroke. METHODS AND RESULTS: The associations amongst B-mode ultrasound determined common carotid IMT, carotid plaque (focal IMT > 1.2 mm) and carotid stenosis (lumen reduction of >15%) and incident CE, were investigated in relation to cardiovascular risk factor levels. Age- and sex-adjusted common carotid IMT, carotid plaque and carotid stenosis were significantly (P < 0.05) related to future CE. Adjustment for established risk factors generally reduced the hazard rate ratios. However, the continuous measure of common carotid IMT, carotid plaque and carotid stenosis were significantly related to incident CE, even after risk factor adjustment. The strength of the associations between common carotid IMT and CE was only to a small extent reduced after adjustment for presence of carotid plaque. There were no statistically significant associations between common carotid IMT, carotid plaque or carotid stenosis and short-term case-fatality rates (28-days mortality) or long-term case-fatality rates (5-years mortality). CONCLUSIONS: The results show an association between common carotid IMT and incident CE, independently of cardiovascular risk factors and carotid plaque. However, there was no association with short-term or long-term mortality after a CE.

Dietary habits after myocardial infarction - results from a cross-sectional study.

OBJECTIVE: Comparing habitual nutrient intakes in persons with a history of acute myocardial infarction (AMI), and age-matched controls. Design. Cross-sectional study. Subjects. Men and women (525 cases and 1890 matched controls), aged 47-73 years, of the population-based Malmö Diet and Cancer cohort. METHODS: Nutrient intakes were assessed by a validated modified diet history method. Body fatness was assessed by bioimpedance analysis. Case ascertainment was provided by national and regional registries. Men and women were analysed separately. Median time since AMI was 5.5 years in men and 3.8 years in women. Cases reported lower energy intakes (EIs) than controls, despite having similar basal metabolic rates. After adjustment for total EI, both male and female cases had lower fat intake and higher intake of several micronutrients, such as ascorbic acid, folate, and vitamin E, than controls, the difference being largest in men. Most of the cases reporting dietary change quoted 'disease' as their main reason for change. They had lower EI and lower energy-adjusted intake of fat than other cases. CONCLUSIONS: Survivors of AMI reported dietary habits more in line with current recommendations, particularly those who afterwards reported having changed their dietary habits. The possible bias introduced by social desirability is discussed.

Incidence of stroke is related to carotid IMT even in the absence of plaque.

BACKGROUND: The carotid intima-media thickness (IMT) has been associated with incidence of stroke. Whether this association is independent of carotid plaque is controversial. METHODS AND RESULTS: The associations among B-mode ultrasound determined common carotid IMT, carotid plaque (focal IMT>1.2mm) and incident stroke, were investigated in 5163 Swedish middle-aged men and women over a median follow-up of 7 years. Age and sex-adjusted carotid IMT, and carotid plaque were significantly (p<0.05) related to future stroke. Adjustment for cardiovascular risk factors generally reduced the hazard rate ratios, however more prominently so with regard to the carotid measure of plaque than with IMT. The associations between carotid IMT and stroke remained after adjustment for presence of carotid plaque, and graded associations between carotid IMT and stroke was found both among those with and without carotid plaque. CONCLUSIONS: In this population-based study, common carotid IMT was associated with incidence of stroke. This relation was independent of presence of carotid plaque.

Prevalence and prognostic significance of asymptomatic peripheral arterial disease in 68-year-old men with diabetes. Results from the population study 'Men born in 1914' from Malmö, Sweden.

OBJECTIVE: To assess the prevalence of asymptomatic peripheral arterial disease (PAD) in older men with diabetes and to compare the incidence of cardiac events and deaths in diabetic and non-diabetic men with abnormal and normal systolic ankle-brachial pressure index, respectively. RESEARCH DESIGN AND METHODS: Population-based cohort of 68-year-old men (n = 474). Diabetes was defined as history of diabetes or a fasting blood glucose > or = 6.1 mmol/l. PAD was defined as an ankle-brachial pressure index (ABI) < 0.9 in either leg. Fourteen-year mortality and cardiac event rates were based on record linkage with regional and national registers. RESULTS: The prevalence of PAD in men with and without diabetes was 29 and 12%, respectively (p = 0.003). The incidence of cardiac events was 22.9/1000 person years in men free from both diabetes and PAD. In the absence of an abnormal pressure index, diabetes was associated with an event rate of 28.4 (p = 0.469). In the presence of an abnormal index the incidence was 102 (p < 0.001). This pattern remained in the multivariate analysis when other atherosclerotic risk factors were taken into account. Cardiovascular mortality rates similarly differed substantially between diabetic men with and without PAD. CONCLUSIONS: A fasting blood glucose value above 6.1 mmol/l even in the absence of symptoms indicating diabetes was associated by an increased prevalence of asymptomatic PAD. The cardiovascular risk in diabetes varied widely between men with and without abnormal ankle-brachial pressure index.

Risk of myocardial infarction and stroke in smokers is related to plasma levels of inflammation-sensitive proteins.

BACKGROUND: The extent to which differences in cardiovascular risk between smokers with similar daily tobacco consumption may be related to plasma levels of inflammation-sensitive proteins (ISP) and whether these proteins are associated with levels of carboxyhemoglobin (COHb%) have not been clarified. METHODS AND RESULTS: In a population-based cohort of 1489 never smokers, 1685 former smokers, and 2901 current smokers, aged 28 to 61 years, plasma levels of orosomucoid (alpha(1)-acid glycoprotein), alpha(1)-antitrypsin, haptoglobin, fibrinogen, and ceruloplasmin were measured. COHb% levels were available for 2098 of them. Incidence of myocardial infarction, stroke, and death were monitored over 18.7+/-4.7 years. The proportion with high ISP levels (ie, > or =2 ISP in the top quartile) increased progressively with daily tobacco consumption (P<0.01) and COHb% (P<0.01). In all smoking categories, the incidence of stroke, cardiac events, and death was related to ISP. In heavy smokers, high ISP levels were associated with adjusted relative risks of 1.57 (1.05 to 2.35) and 1.50 (1.11 to 2.03) for cardiac events and death, respectively. Corresponding figures for moderate and light smokers were 1.59 (1.13 to 2.24) and 1.14 (0.87 to 1.49), respectively, and 1.32 (0.95 to 1.85) and 1.48 (1.10 to 1.98), respectively. CONCLUSIONS: ISP levels are related to COHb% in smokers. High levels are associated with an increased cardiovascular risk.

Leg blood flow and long-term cardiovascular prognosis in men with typical and atypical intermittent claudication.

OBJECTIVES: To compare leg blood flow and the long-term cardiovascular prognosis in men with typical and atypical intermittent claudication (IC) according to Rose's questionnaire. METHODS: Leg blood flow during reactive hyperaemia was assessed at 55 years of age by calf plethysmography. Measurement of the systolic ankle-arm pressure index was used to assess the prevalence of peripheral arterial disease (PAD) at 68 years of age. Mortality and incidence of cardiac events is based on record linkage with regional and national registers. RESULTS: Twenty-one (3%) out of 700 men had typical IC at 55 years of age. Sixteen (76%) of these had normal plethysmography, with a mean peak flow (95% CI) of 24.6 (19.3-30.0) ml/min/100 ml. Peak flows were similar and normal in men with atypical IC (mean 22.5; 95% CI 21.3-23.6) and men having no pain (mean 23.8; 95% CI 23.1-24.4). Mortality rates were increased in men with typical IC but who had normal leg blood flow (49.5 deaths/1000 person years; p = 0.008), and men having atypical IC (35.3 deaths/1000 person years; p = 0.007) in comparison with men having no leg pain (27.4 deaths/1000 person years). This could not be accounted for by an increased cardiac event rate. The prevalence of PAD at 68 years of age was not increased in either of these two groups. CONCLUSIONS: Only 12% of subjects with exertional pain fulfilled all criteria for typical IC. Typical and atypical claudication in absence of objective evidence of PAD was associated with a reduced life expectancy, but not with an increased cardiovascular risk. To properly assess the prognosis, non-invasive assessment of PAD should be used in addition to questionnaires.