Cardiovascular Inflammatory Markers Research Articles

Circulating Galectin-3 in Patients with Cardiogenic Shock Complicating Acute Myocardial Infarction Treated with Mild Hypothermia: A Biomarker Sub-Study of the SHOCK-COOL Trial.

Background: Galectin-3 (Gal-3) is considered a potential cardiovascular inflammatory marker that may provide additional risk stratification for patients with acute heart failure. It is unknown whether mild therapeutic hypothermia (MTH) impacts Gal-3 levels. Therefore, this biomarker study aimed to investigate the effect of MTH on Gal-3. Methods: In the randomized SHOCK-COOL trial, 40 patients with cardiogenic shock (CS) complicating acute myocardial infraction (AMI) were randomly assigned to the MTH (33 °C) or control group in a 1:1 ratio. Blood samples were collected on the day of admission/day 1, day 2, and day 3. Gal-3 level kinetics throughout these time points were compared between the MTH and control groups. Additionally, potential correlations between Gal-3 and clinical patient characteristics were assessed. Multiple imputations were performed to account for missing data. Results: In the control group, Gal-3 levels were significantly lower on day 3 than on day 1 (day 1 vs. day 3: 3.84 [IQR 2.04−13.3] vs. 1.79 [IQR 1.23−3.50] ng/mL; p = 0.049). Gal-3 levels were not significantly different on any day between the MTH and control groups (p for interaction = 0.242). Spearman’s rank correlation test showed no significant correlation between Gal-3 levels and sex, age, smoking, body mass index (BMI), and levels of creatine kinase-MB, creatine kinase, C-reactive protein, creatinine, and white blood cell counts (all p > 0.05). Patients with lower Gal-3 levels on the first day after admission demonstrated a higher risk of all-cause mortality at 30 days (hazard ratio, 2.67; 95% CI, 1.11−6.42; p = 0.029). In addition, Gal-3 levels on day 1 had a good predictive value for 30-day all-cause mortality with an area under the receiver operating characteristic curve of 0.696 (95% CI: 0.513−0.879), with an optimal cut-off point of less than 3651 pg/mL. Conclusions: MTH has no effect on Gal-3 levels in patients with CS complicating AMI compared to the control group. In addition, Gal-3 is a relatively stable biomarker, independent of age, sex, and BMI, and Gal-3 levels at admission might predict the risk of 30-day all-cause mortality.

Does reproductive stage impact cardiovascular disease risk factors? Results from a population-based cohort in Lausanne (CoLaus study).

Menopause has been associated with adverse cardiovascular disease (CVD) risk profile, yet it is unclear whether the changes in CVD risk factors differ by reproductive stage independently of underlying ageing trajectories. The CoLaus study is a prospective population-based cohort study in Lausanne, Switzerland. We used data from women at baseline and follow-up (mean: 5.6 ± 0.5 years) from 2003 to 2012 who did not use hormone therapy. We classified women into (i) premenopausal, (ii) menopausal transition, (iii) early (≤5 years)and (iv) late (>5 years) postmenopausal by comparing their menstruation status at baseline and follow-up. We measured fasting lipids, glucoseand cardiovascular inflammatory markers. We used repeated measures (linear mixed models) for longitudinal analysis, using premenopausal women as a reference category. We adjusted analyses for age, medicationsand lifestyle factors. We used the data from 1710 women aged 35-75 years. Longitudinal analysis showed that the changes in CVD risk factors were not different in the other three menopausal categories compared to premenopausal women. When age was used as a predictor variable and adjusted for menopause status, most CVD risk factors increased, while interleukin-6 and interleukin-1β decreased with advancing age. The current study suggests that women have a worsening cardiovascular risk profile as they age, and although menopausal women may have higher levels of cardiovascular risk factors compared to premenopausal women at any given time, the 5-year changes in cardiovascular risk factors may not depend on the reproductive stage.

Positive effects of renal denervation on markers of cardiovascular inflammation and left ventricular mass. 24-months follow-up

Aim. To study the long-term effect of renal denervation (RDN) on left ventricular mass (LVM) and inflammatory markers in resistant hypertensive patients.Material and methods. Forty-one patients with resistant hypertension and 24-h blood pressure (BP) 158,7±15,8/87,3+14,6 mmHg, aged 56,6+10,2 years, were enrolled in the study and undergone RDN. Mean 24-h BP, left ventricular mass (transthoracic echocardiography), high sensitivity C-reactive protein (hsCRP), interleukin- 1β (IL­1β), IL-6, IL-10) and tumor necrosis factor alpha (TNF- α) were assessed at baseline and 2 years after the RDN.Results. A baseline prevalence of left ventricular hypertrophy (LVH) was 90,2%. Two years after RDN LVM and interventricular septum (IVS) decreased significantly (p<0.05 for both). Decrease in myocardial mass (∆LVM >0 g) was documented in 24 patients. The regression of LVM was accompanied by a significant decrease in levels of inflammatory markers — hsCRP by 38,3% (p=0,031), TNF-α by 60,7% (p=0,009), IL- 1β — by 71,1% (p=0,001), and IL-10 by 58,2% (p=0,001). In patients in the absence of LVM regression only TNF-α decreased significantly (-68,8%, p=0,001). There was no correlation between changes of LVM and the inflammatory markers at 24 months after RDN.Conclusion. The RDN in RH patients may have long-term cardioprotective effect in terms of significant regress of LVH, which may be partly attributed to the regress in systemic or myocardial inflammation.

Personal informatics and negative emotions during commuter driving: Effects of data visualization on cardiovascular reactivity & mood

Mobile technology and wearable sensors can provide objective measures of psychological stress in everyday life. Data from sensors can be visualized and viewed by the user to increase self-awareness and promote adaptive coping strategies. A capacity to effectively self-regulate negative emotion can mitigate the biological process of inflammation, which has implications for long-term health. Two studies were undertaken utilizing a mobile lifelogging platform to collect cardiovascular data over a week of real-life commuter driving. The first was designed to establish a link between cardiovascular markers of inflammation and the experience of anger during commuter driving in the real world. Results indicated that an ensemble classification model provided an accuracy rate of 73.12% for the binary classification of episodes of high vs. low anger based upon a combination of features derived from driving (e.g. vehicle speed) and cardiovascular psychophysiology (heart rate, heart rate variability, pulse transit time). During the second study, participants interacted with an interactive, geolocated visualisation of vehicle parameters, photographs and cardiovascular psychophysiology collected over two days of commuter driving (pre-test). Data were subsequently collected over two days of driving following their interaction with the dynamic, data visualization (post-test). A comparison of pre- and post-test data revealed that heart rate significantly reduced during episodes of journey impedance after interaction with the data visualization. There was also evidence that heart rate variability increased during the post-test phase, suggesting greater vagal activation and adaptive coping. Subjective mood data were collected before and after each journey, but no statistically significant differences were observed between pre- and post-test periods. The implications of both studies for ambulatory monitoring, user interaction and the capacity of personal informatics to enhance long-term health are discussed.

160-LB: Effect of Sodium-Glucose Cotransporter 2 Inhibitors on Hemodynamic and Metabolic Cardiovascular Markers in Patients with Type 2 Diabetes Mellitus

Background: Sodium glucose co-transporter 2 (SGLT2) inhibitors could early reduce cardiovascular (CV) and heart failure (HF) risk in patients with type 2 diabetes mellitus (T2DM) in recent CV outcome trials. We hypothesized that benefit of SGLT2 inhibitors would be early on both hemodynamic and metabolic CV markers. Objectives: The goal of this study was to examine the effects of empagliflozin or dapagliflozin on CV markers. Factors associated beneficial CV marker changes were determined. Material and Method: This prospective study included 60 patients with T2DM, In ASCVD group (n = 18), all patients received empagliflozin (n = 18). In non-ASCVD groups (n = 42), they were allocated to received either empagliflozin or dapagliflozin. The study assessed the change in serum N-terminal pro-B-type natriuretic peptide (NT-proBNP), apolipoprotein B (apo B), and highly sensitive C-reactive protein (hsCRP) from baseline to 4, and 26 weeks. Results: Median change of NT-proBNP from baseline after receiving SGLT2 inhibitors were -11.1 (-30.4, -1.3) pg/mL, and -11.5 (-30.4, -1.5) pg/mL at weeks 4, and 26, respectively (all p <0.05). Although, median change of body weight (BW) were initially -0.77 (-1.11, -0.44) kg and -1.65 (-2.3, -1.01) kg at the end of the study, both apo B and hsCRP were not statistically changed. We found that baseline NT-proBNP >125 pg/mL could predict beneficial in hemodynamic in CV marker changes with sensitivity 58.3%, specificity 96.6%, positive predictive value 87.5%, negative predictive value 84.8% and accuracy 85.4%. Conclusion: We demonstrated that SGLT2 inhibitors had early beneficial effect in hemodynamic CV marker. Furthermore, SGLT2 inhibitors didn’t have detrimental effect on both atherogenic lipoprotein particle and inflammatory CV markers. Measurement of NT-proBNP level might enhance CV risk stratification and guide introduction of SGLT2 inhibitors in both ASCVD and non-ASCVD patients with T2DM. Disclosure N. Sathavarodom: None.

A 6-Week Worksite Positivity Program Leads to Greater Life Satisfaction, Decreased Inflammation, and a Greater Number of Employees With A1C Levels in Range.

To determine whether a 6-week Positivity Program could impact employee cardiovascular inflammation, blood sugars, cortisol, dehydroepiandrosterone (DHEA), and/or life satisfaction. Pre- and post-study blood draw and life satisfaction questionnaire tracked changes in 10 cardiovascular and inflammatory biomarkers for 63 employees who participated in a 6-week Positivity Program comprised of three interventions: gratitude, HeartMath's Heart Lock-In, and yoga stretches with guided imagery. Improvements were recorded in life satisfaction as well as in seven of 10 cardiovascular and inflammatory biomarkers, including high sensitivity C-reactive protein (HsCRP) (-27%), hemoglobin A1c (HbA1c) (-1%), glucose (-2%), myeloperoxidase (MPO) (-5%), lipoprotein-associated phospholipase-A2 (Lp-PLA2) (-9%), apolipoprotein B (ApoB) (-6%), and DHEA (1%). No improvements were recorded in cortisol (11%), small-dense LDL (sdLDL) (0%), or oxidized LDL (OxLDL) (7%). Data suggest that engaging in 6 weeks of a workplace Positivity Program may improve employee life satisfaction, blood sugar levels, and some markers of cardiovascular inflammation.

Cardiovascular markers of inflammation and serum lipid levels in HIV-infected patients with undetectable viremia

Patients successfully treated for HIV infection still have an increased risk for cardiovascular morbidity and mortality, which might be related not only to traditional risks, but also to inflammation and dyslipidemia. We examined the relationship of serum lipid levels with plasma biomarkers of inflammation using a composite inflammatory burden score (IBS) based on individual (>75th percentile) measurements from the following seven markers: CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin. IBS was categorized as 0 (none of the biomarkers >75th percentile), 1, 2 and 3 or more scores. Correlations between the IBS and lipid parameters were examined by ordered logistic regression proportional odds models to estimate the odds of more elevated biomarkers. 181 male patients with undetectable HIV-viremia were included into the study. In the multivariate model, a one-unit increase (mmol/L) of total cholesterol and triglycerides was associated with a 1.41-fold (95% CI, 1.13–1.76) and 1.37-fold (95% CI, 1.18–1.60) increased odds of having a greater IBS, respectively. Those with an IBS score ≥1 compared to none had 2.14 (95% CI, 1.43–3.20) higher odds of having a one-unit increased total cholesterol/HDL-cholesterol ratio. In successfully treated HIV-infected persons dyslipidemia was associated with inflammation.

Resveratrol protects against diet-induced atherosclerosis by reducing low-density lipoprotein cholesterol and inhibiting inflammation in apolipoprotein E-deficient mice.

Resveratrol (RES) is a polyphenol compound that has been shown a promising cardioprotective effect. However, some reports have yielded conflicting findings. Herein, we investigated the anti-atherosclerotic effects of RES in apolipoprotein E (apo E)-deficient mice on a high cholesterol diet. Firstly, atherosclerosis was induced by feeding a high cholesterol diet to apo E-deficient mice. Then, we examined its effects on weight control, and serum interleukin-6 (IL-6) levels and used histopathological methods to analyze morphology and inflammatory marker of atherosclerotic lesions in mice orally supplemented with high (25 mg/kg/day) and low (5 mg/kg/day) doses of RES for 8 weeks. Mice with high dose of RES had reduced epididymal fat pads, and lower serum IL-6 levels compared with those of control mice. Moreover, RES in high doses also decreased the low-density lipoprotein cholesterol (LDL-C) levels and atherogenic index (LDL-C/HDL-C) in the mice. Dissection of high-dose RES-treated mice revealed a marked reduction in fat deposition, percentage of mice with atherosclerotic lesion, and intima/media ratio in the aortic areas. The expressions of macrophage-specific marker F4/80 and cardiovascular inflammatory marker NF-κB in atherosclerotic vessels were both diminished in the atherosclerotic vessels of high-dose RES-supplementated apo E-deficient mice. These results suggest that RES prevented the effects of a high cholesterol diet on the rate of accretion in atherosclerosis progression by reducing the LDL-C levels and suppressing atherosclerotic inflammation. RES can therefore be valuable in the development of new anti-atherosclerotic agents.

Cardiovascular markers of inflammation and serum lipid levels in HIV-infected patients with undetectable viraemia.

IntroductionSuccessfully treated HIV-infected patients may still have an increased risk for cardiovascular morbidity and mortality, which might be related not only to traditional risks, but also to inflammation and dyslipidemia induced by HIV and/or antiretroviral therapy [1, 2]. We examined the relationship of serum lipid levels with plasma biomarkers of inflammation using a composite inflammatory burden score (IBS) from the following seven markers of inflammation: CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin.Materials and MethodsSubjects were selected among consecutive HIV-infected males ≥18 years of age with an undetectable viral load (<50 copies/mL of HIV1-RNA), seen at the University Hospital for Infectious Diseases, Zagreb, Croatia, in the period from January 2012 to March 2013. Plasma inflammatory biomarkers (CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin, quantified by bead-based cytometry) >75th percentile were considered elevated and an IBS was constructed as the presence of zero, one, two, or three or more elevated biomarkers. Correlations between the IBS and lipid parameters were examined using Spearman's Rho and by ordered logistic regression proportional odds model to estimate the odds of more elevated (>75th percentile) biomarkers.Results181 male patients were included into the study, the median age was 46.7 (Q1–Q3, 39.9–55.0) years and the median current CD4 cell count was 553.0 (Q1–Q3, 389–729) per microliter. The patients were mainly treated with two nucleoside reverse transcriptase inhibitor (NRTI) plus one non-NRTI (NNRTI) (N=100, 60.8%) or two NRTI plus lopinavir (N=50, 27.6%). There was a significant correlation between the IBS and serum cholesterol (Rho=0.23, 95% CI, 0.09–0.37), triglycerides (Rho=0.30, 95% CI, 0.16–0.42) and cholesterol/HDL-cholesterol ratio (Rho=0.25, 95% CI 0.11–0.38). In the multivariable model a one unit increase in cholesterol/HDL-cholesterol ratio was associated with a 1.72-fold (95% CI, 1.27–2.33) increased odds of having a greater IBS. One unit increase (mmol/L) of cholesterol and triglycerides was associated with a 1.41-fold (95% CI, 1.13–1.76) and 1.37-fold (95% CI, 1.18–1.60) increased odds of having a greater IBS, respectively.ConclusionsOur study suggests that in virologically suppressed patients there is a significant association between markers of inflammation and serum levels of cholesterol and triglycerides as well as the cholesterol/HDL-cholesterol ratio.

Periodontal Status Affects C‐Reactive Protein and Lipids in Patients With Stable Heart Disease From a Tertiary Care Cardiovascular Clinic

There are scarce data on the impact of the periodontal condition in the control of biomarkers in patients with cardiovascular disease (CVD). The aim of this study is to assess whether periodontal inflammation and tissue breakdown are associated with C-reactive protein (CRP) and lipids in patients with stable heart disease. This cross-sectional study included 93 patients with stable coronary artery disease (57 males; mean age: 63.5 ± 9.8 years) who were in outpatient care for at least 6 months. After applying a structured questionnaire, periodontal examinations were performed by two calibrated periodontists in six sites per tooth at all teeth. Blood samples were collected from patients on the day of periodontal examination to determine levels of CRP, lipids, and glycated hemoglobin. Multiple linear regression models were fitted to evaluate the association among different periodontal and blood parameters controlling for sex, body mass index, glycated hemoglobin, use of oral hypoglycemic drugs, and smoking. Overall, the sample presented high levels of periodontal inflammation and tissue breakdown. Unadjusted mean concentrations of triglycerides (TGs), very-low-density lipoprotein cholesterol, and glucose were significantly higher in individuals with severe periodontitis. When multiple linear regression models were applied, number of teeth with clinical attachment loss ≥6 mm and presence of severe periodontitis were significantly associated with higher CRP concentrations. Bleeding on probing was significantly associated with TGs, total cholesterol, and non-high-density lipoprotein cholesterol. In this sample of patients with stable CVD, current periodontal inflammation and tissue breakdown are associated with cardiovascular inflammatory markers, such as CRP and lipid profile.

Elevated high sensitivity C-reactive protein levels in aging men with low testosterone

Objective. We examined baseline data from a lipid treatment study to assess the relationship between testosterone (T) and the cardiovascular inflammatory marker, high sensitivity C-reactive protein (hsCRP).Methods. The baseline T, hsCRP, lipid, glycemic, and anthropometric data were obtained from 467 men (mean age: 52 years). Inclusion criteria included low-density lipoprotein cholesterol ≥ 3.4 to 4.9 mmol/l and triglycerides ≤ 4.0 mmol/l. The baseline hsCRP levels were examined across the following T subgroups: <6.9 nmol/l (moderate to severe hypogonadism), 6.9 to <10.4 nmol/l (mild to moderate hypogonadism), 10.4 to <15 nmol/l (low-normal T), and ≥ 15 nmol/l (normal T).Results. The median hsCRP levels were significantly (p = 0.041) different across the four T subgroups; patients in the lower T subgroups had higher median hsCRP levels than patients in the higher T subgroups. The percentage of men with elevated hsCRP (>2 mg/l) was also significantly (p = 0.038) different across the four T subgroups; 83% of men with T < 6.9 nmol/l had elevated hsCRP compared with 40% with T ≥ 15 nmol/l.Conclusions. This analysis demonstrated an inverse relationship between serum T and hsCRP in aging men. Urologists need to be aware that low T levels may not only adversely affect sexual function but also may worsen cardiovascular risk in aging, hypogonadal men.

Effect of pioglitazone on insulin sensitivity, vascular function and cardiovascular inflammatory markers in insulin‐resistant non‐diabetic Asian Indians

To determine the effects of pioglitazone (30 mg once daily for 16 weeks) on insulin sensitivity, insulin-mediated vasodilation, vascular inflammatory markers, fat distribution and lipids in Asian Indians and Caucasians of European ancestry. Cross-sectional study. Eighteen non-diabetic Asian Indians and 17 Caucasians of comparable age (34 +/- 3 vs. 36 +/- 3 years) and body mass index (26.0 +/- 1.2 vs. 24.7 +/- 1.0 kg/m(2)) had measurements of insulin sensitivity (M, insulin clamp at 6 pmol/kg per min), abdominal fat (computed tomographic scan at L4-L5), endothelial-dependent (reactive hyperaemia, RH) and -independent (0.4 mg sublingual nitroglycerin, TNG) vasodilation using brachial artery ultrasound before and after the 2-h clamp at baseline and after pioglitazone therapy. Asian Indians were insulin resistant compared with Causasians during the baseline clamp (M = 25.6 +/- 1.7 vs. 41.1 +/- 2.2 micromol/kg per min, P < 0.0001) and improved significantly after pioglitazone (to 33.9 +/- 1.7 micromol/kg per min, P < 0.001). Vasodilatory responses to RH and TNG were similar in Asian Indians and Caucasians at baseline and did not change. Insulin-mediated vasodilation improved after pioglitazone in Asian Indians, but not in Caucasians, and correlated with the change in insulin sensitivity (r = 0.52, P = 0.03). C-reactive protein (CRP) was higher in Asian Indians vs. Caucasians (1.6 +/- 0.4 vs. 0.9 +/- 0.2 mg/l) and was negatively correlated with insulin sensitivity (r = -0.53, P = 0.02). In the Asian Indian group, CRP and plasminogen activator inhibitor-1 decreased and adiponectin increased after pioglitazone, but there were no significant changes in total or visceral fat. These results demonstrate that insulin-resistant Asian Indians respond favourably to an insulin sensitizer with improvements in insulin sensitivity, cardiovascular and inflammatory risk markers, and vascular responses to insulin. These agents may have a role in decreasing the risk of diabetes and cardiovascular disease in this high-risk population.

Effect of vitamin E supplementation with and without hormone therapy on circulatory inflammatory markers in postmenopausal women

To investigate the effect of vitamin E with and without estrogen replacement therapy or hormone therapy (HT) on inflammatory markers in postmenopausal women. Prospective, observational study, followed by a randomized, prospective, double-blind study. Healthy volunteers in an academic medical referral center in Dallas, Texas. Seventy-five postmenopausal healthy women, ages 40 to 65 years, with follicle-stimulating hormone (FSH) levels greater > or =30 mIU/mL and a serum estradiol level < or =30 pg/mL. After enrollment, all women were studied at baseline and received vitamin E for 4 weeks. They were then randomized from week 4 to week 12 to receive vitamin E in conjunction with conjugated equine estrogen (CEE) (0.625 mg), CEE (0.625 mg) plus medroxyprogesterone acetate (MPA) (2.5 mg), or placebo. Change from baseline and between groups effects of vitamin E with and without estrogen or hormone therapy on seven circulatory inflammatory markers in postmenopausal women. Vitamin E levels increased to a similar extent in all three groups compared with baseline at weeks 4 and 12. Vitamin E increased serum interleukin-6 levels. Combination CEE plus MPA significantly increased C-reactive protein levels. However, there were no consistent statistically significant effects on six other inflammatory markers. Vitamin E attenuated C-reactive protein increases in postmenopausal women treated with estrogen replacement therapy but not with HT. Because there was no other persistent effect on six additional inflammatory markers, it can be concluded that vitamin E and HT do not play a major role in promoting a change in cardiovascular inflammatory markers.

We-P11:159 Hypocaloric diet rapidly and significantly reduces in short term cardiovascular inflammatory markers in overweight and obese patients

We-P11:159 Hypocaloric diet rapidly and significantly reduces in short term cardiovascular inflammatory markers in overweight and obese patients

The Effect of Regular Exercise on Cardiovascular Inflammation Markers in Elderly Korean Men

This study was performed to investigate the effect of regular aerobic exercise on the representative markers of cardiovascular inflammatory diseases. Biomarkers of cardiovascular disease were measured in 89 healthy elderly males (age range; 47–88) living in Seoul, Korea. Questionnaires were used to assess the status of the regular exercise participants. Various factors associated with chronic cardiovascular diseases, including total cholesterol, triglyceride, phospholipid, platelet, and C-reactive protein were lowered in regular exercise participants (p<0.05). In contrast, serum albumin concentration was higher in the exercised group compared with a non-exercised group (p<0.05). Therefore it was concluded that participation in regular exercise reduces cardiovascular inflammation and offers protection against chronic cardiovascular disease in elderly Korean men.

The relationship between attendance at religious services and cardiovascular inflammatory markers.

Previous studies have shown an association between attendance at religious services and health, particularly cardiovascular morbidity and mortality. Little research has focused on religious attendance and physiological markers of cardiovascular risk. The purpose of this study was to explore the relationship between religious attendance and inflammatory markers of cardiovascular risk. Nationally representative sample of non-institutionalized United States adults aged 40 and over derived from the National Health and Nutrition Examination Survey III 1988-1994 (n = 10,059). The main outcome measures were the inflammatory system markers C-reactive protein, fibrinogen, and white blood cell count. 40.8 percent of the population attended religious services 40 or more times in the previous year while 22.4 percent attended services less than 40 times and 36.8 percent attended no religious services at all. Non-attenders of religious services were more likely than attenders to have elevated white blood cell counts (p = .001), highly elevated C-reactive protein (p = .02), and elevated fibrinogen (p = .05). After adjusting for demographic variables, health status, and BMI, the association between religious attendance and cardiovascular markers remained. Once current smoking was added to the model the independent effect of religious attendance dropped below conventional confidence limits. These findings suggest that people who have attended religious services in the previous year are less likely to have elevated levels of certain inflammatory markers, however, current smoking has significant shared variance with religious attendance.