Daniela Zahn

Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

Social isolation and depression predict 12-month outcomes in the “waiting for a new heart study”

BACKGROUND Identification of modifiable psychosocial characteristics related to survival of heart transplant (HTx) candidates is needed to prevent clinical deterioration and improve prognosis. METHODS A multi-site, prospective study was conducted with 318 HTx candidates (18% female, 82% male; 53 +/- 11 years of age) newly listed at 17 hospitals in Germany and Austria. Baseline demographic and psychosocial characteristics were assessed by questionnaires. Indicators of disease severity (Heart Failure Survival Score, creatinine, cardiac index) and 12-month outcomes (death, high-urgency HTx, elective HTx, de-listing due to deterioration or improvement) were provided by Eurotransplant. RESULTS By 12 months, 33 patients died, 83 received an urgent HTx, 30 underwent an elective HTx, and 9 were de-listed due to clinical deterioration and 17 due to improvement. All measures of disease severity predicted outcomes. Controlling for disease severity, the number of social contacts contributed significantly to outcomes, favoring those who improved. Comparing socially isolated patients (<4 social contacts/month) who also had depression scores in the clinical range (high psychosocial risk group; n = 37) to those with >10 social contacts/month without depression (low psychosocial risk group; n = 47) revealed significant differences in the distribution of outcome frequencies (chi-square = 11.2, df = 4, p < 0.04). The high psychosocial risk group was more likely to have died/deteriorated and less likely to have improved than the low psychosocial risk group. CONCLUSIONS Regardless of disease severity, socially isolated HTx candidates who are also depressed may be at increased risk for clinical deterioration and mortality, indicating a need for psychosocial intervention.

Cognitive Behavioral Therapy Versus Sertraline in Patients With Depression and Poorly Controlled Diabetes: The Diabetes and Depression (DAD) Study: A Randomized Controlled Multicenter Trial.

OBJECTIVE This study compared the long-term efficacy of a diabetes-specific cognitive behavioral group therapy (CBT) with sertraline in patients with diabetes and depression who initially responded to short-term depression treatment. RESEARCH DESIGN AND METHODS A randomized controlled single-blind trial was conducted in 70 secondary care centers across Germany comparing 12 weeks of CBT with sertraline in 251 patients with type 1 or 2 diabetes (mean HbA1c 9.3%, 78 mmol/mol) and major depression (Structured Clinical Interview for DSM-IV [SCID]). After 12 weeks, treatment responders (≥50% reduction Hamilton Depression Rating Scale [HAMD-17]) were included in the 1-year study phase where CBT patients were encouraged to use bibliotherapy and sertraline patients received continuous treatment. We analyzed differences for HbA1c (primary outcome) and reduction (HAMD-17) or remission (SCID) of depression from baseline to the 1-year follow-up using ANCOVA or logistic regression analysis. RESULTS After 12 weeks, 45.8% of patients responded to antidepressant treatment and were included in the 1-year study phase. Adjusted HbA1c mean score changes from baseline to the end of the long-term phase (−0.27, 95% CI −0.62 to 0.08) revealed no significant difference between interventions. Depression improved in both groups, with a significant advantage for sertraline (HAMD-17 change: −2.59, 95% CI 1.15–4.04, P < 0.05). CONCLUSIONS Depression improved under CBT and sertraline in patients with diabetes and depression, with a significant advantage for sertraline, but glycemic control remained unchanged. CBT and sertraline as single treatment are insufficient to treat secondary care diabetes patients with depression and poor glycemic control.

Depression and disease severity as correlates of everyday physical activity in heart transplant candidates.

It is unclear to what extent patients awaiting heart transplantation (HTx) engage in physical activities. We examined the everyday physical activity and its associations with depressive symptoms and disease severity in 318 patients newly registered for HTx in the multi‐site study ‘Waiting for a New Heart’ (aged 53.5 ± 11.4 years, 18% female patients). Participants completed questionnaires assessing depressive symptomatology and physical activity (number of physical activities, caloric expenditure associated with each activity), and estimated the distance they were able to walk without a break. Medical parameters at the time of listing [e.g. peak oxygen consumption (peakVO2); the German Transplant Society Score (GTSS)] were provided by Eurotransplant. Almost 50% of patients engaged in activities of daily living (housework, walking), but <10% engaged in regular exercise. All physical activity measures correlated significantly with peakVO2 (Ps < 0.01). Elevated depression scores were present in 39% of patients. Controlling for confounding variables (e.g. peakVO2, diastolic blood pressure, GTSS, age), depressive symptomatology accounted for additional variance in all physical activity measures (Ps < 0.05). The association of depressive symptoms with reduced physical activity suggests two important perspectives: attempts to increase physical activity (especially in the area of daily living) might benefit from targeting depression, and increased physical activity might also help to reduce depressive symptoms.

Heart rate variability and self-control—A meta-analysis

Heart rate variability (HRV) has been suggested as a biological correlate of self-control. Whereas many studies found a relationship between HRV at rest and self-control, effect sizes vary substantially across studies in magnitude and direction. This meta-analysis evaluated the association between HRV at rest and self-control in laboratory tasks, with a particular focus on the identification of moderating factors (task characteristics, methodological aspects of HRV assessment, demographics). Overall, 24 articles with 26 studies and 132 effects (n=2317, mean age=22.44, range 18.4-57.8) were integrated (random effects model with robust variance estimation). We found a positive average effect of r=0.15, 95% CI [0.088; 0.221], p<0.001 with a moderate heterogeneity (I(2)=56.10%), but observed evidence of publication bias. Meta-regressions did not reveal significant moderators. Due to the presence of potential publication bias, our results have to be interpreted cautiously.

Cortisol, platelet serotonin content, and platelet activity in patients with major depression and type 2 diabetes: an exploratory investigation.

Objective Hypothalamic-pituitary-adrenal system dysfunction, serotonergic system alterations, and enhanced platelet activity may contribute to the increased cardiac risk in depression. This exploratory study examined associations between cortisol parameters, platelet serotonin (5-HT) content, and platelet activity markers in patients with newly diagnosed major depression (MD) and/or Type 2 diabetes (T2DM) compared with healthy controls. Methods We compared cortisol awakening response (CAR), diurnal decrease in salivary cortisol concentrations (slope), platelet 5-HT, and platelet markers (CD40, CD40 ligand [CD40L], soluble CD40L, CD62P, &bgr;-thromboglobulin, and platelet factor-4) in 22 T2DM patients, 20 MD patients, 18 T2DM patients with MD, and 24 healthy controls. Results Platelet markers were elevated in MD (F(6,60) = 11.14, p < .001) and T2DM (F(6,60) = 13.07, p < .001). Subgroups did not differ in 5-HT or cortisol slope, whereas T2DM patients without depression had significantly lower CAR than did healthy controls (F(1,61) = 7.46, p = .008). In healthy controls, cortisol slope correlated with platelet activity for CD40 (r = −0.43, p = .048) and 5-HT was correlated with CD40L (r = 0.53, p = .007). In patients with both T2DM and MD, 5-HT and CD62P were correlated (r = 0.52, p = .033). Conclusions Increased platelet activity in T2DM and MD may play a role in the association between diabetes, depression, and coronary artery disease. The present data suggest that group differences in cortisol or 5-HT as well as group-specific associations of cortisol or 5-HT with platelet markers might be of limited importance in the shared pathways of T2DM and depression in the pathophysiology of coronary artery disease.

Composite risk scores and depression as predictors of competing waiting‐list outcomes: the Waiting for a New Heart Study

We evaluated two composite risk scores, (Heart Failure Survival Score, HFSS; German Transplant Society Score, GTSS), and depression as predictors of mortality and competing waiting‐list outcomes [high‐urgency transplantation (HU‐HTx), elective transplantation, delisting because of clinical improvement] in 318 heart transplant (HTx) candidates (18% women; aged 53 ± 11 years) from 17 hospitals and newly registered with Eurotransplant. Demographic variables and depression (Hospital Anxiety and Depression Scale, HADS) were assessed using questionnaires. Variables to compute HFSS and GTSS, age, medications, and outcomes were provided by Eurotransplant. At 12 months, 33 patients died, 83 received urgent HTx, 30 elective HTx, and 17 were delisted because of improvement. Applying cause‐specific Cox regressions, only the HFSS was significantly associated with 1‐year mortality [HR = 0.64 (95% CI = 0.43–0.95), P = 0.029]. The GTSS was the strongest predictor of HU‐HTx [HR = 1.02 (95% CI = 1.01–1.02), P < 0.001]. Low depression scores contributed significantly to clinical improvement, even after adjusting for age and risk scores [HADS: HR = 0.12 (95% CI = 0.02–0.89), P = 0.039]. These findings confirm the usefulness of composite risk scores for the prediction of mortality and HU‐HTx, validating both scores for their intended use. The finding that depression was an independent predictor of the waiting‐list outcome clinical improvement suggests that considering patients’ psychological attributes in addition to their medical characteristics is advisable.

New pathways of increased cardiovascular risk in depression: a pilot study on the association of high-sensitivity C-reactive protein with pro-atherosclerotic markers in patients with depression.

BACKGROUND An elevation of inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP) can be found in patients with depressive disorders. Inflammatory processes are known to influence atherosclerosis and might also mediate the link between depression and diabetes. The present study aimed at comparing hs-CRP and its relationship with atherogenic platelet markers in patients with type 2 diabetes (TD2) and/or newly diagnosed major depression (MD). METHODS Hs-CRP concentrations in 24 patients with TD2, 21 patients with MD (diagnosed according to ICD-10 and DSM-IV), 19 patients with TD2 and comorbid MD, and 25 healthy controls were compared using analysis of variance. The relationship of hs-CRP with atherogenic platelet markers (CD40, CD40 ligand, soluble CD40L) were examined for the different samples using Pearsons correlations and regression analyses. RESULTS Hs-CRP levels were not associated with depression (F(1, 80)=0.56, p=.814). There was a trend for higher hs-CRP in diabetes patients (p=.095), but not after adjustment for BMI. CD40 or sCD40L were not related to hs-CRP. For CD40L, regression analysis revealed a significant interaction between hs-CRP and subgroup: Hs-CRP was positively associated with CD40L only in depressed patients without diabetes (B=.334, p<.05). LIMITATIONS Causal inferences are limited because of the cross-sectional design and the small sample size. CONCLUSIONS Our results demonstrate preliminary evidence that hs-CRP might contribute to the risk of cardiovascular disease in depressed patients without somatic diseases via its association with platelet expression of CD40L. Further studies are necessary to confirm these findings.

Patients' sex and emotional support as predictors of death and clinical deterioration in the Waiting for a New Heart Study: results from the 1-year follow-up

Context Little is known about the role of patients sex and emotional support in the prognosis of heart transplant candidates. Objective To examine patients sex and emotional support as predictors of outcomes in the Waiting for a New Heart Study. Design, Setting, and Participants The Waiting for a New Heart Study is a prospective observational study of 318 patients (18% female) newly added to the waiting list for a heart transplant. Demographic, medical, psychosocial characteristics (including social support [ENRICHD Social Support Index; high vs low support]) were assessed at the time of wait-listing. Main Outcomes Time until death/delisting due to deteriorated health, considering competing outcomes (eg, transplantation) during the first 12 months after wait-listing were analyzed via cause-specific Cox proportional hazard models. Results—By 12 months, 32 men (12%) and 10 women (17%) had died/deteriorated. Medical risk was comparable across sexes. More men than women reported low emotional support (20.4% vs 8.6%) and being a past or current smoker (80.4% vs 56.9%). More women than men had low vocational level (93.1% vs 69.6%; all P values < .05). With medical risk and other confounding variables controlled for, female sex significantly increased risk of death/deterioration (hazard ratio, 2.30; 95% confidence interval, 1.04–5.12; P = .04); low emotional support further tended to increase the risk for this outcome (P = .07). As none of the 5 women with low emotional support had reached this end point, analyses were performed in the male sample and revealed that men with low emotional support were more than twice as likely to die/deteriorate than were men with high support (hazard ratio, 2.23; 95% confidence interval, 1.04–4.82; P = .04). Conclusion Women had worse survival while awaiting a heart transplant than men had, independent of confounding variables. Even though emotional support may be an important buffer for men, protective factors for women warrant further investigation with larger samples and/or longer follow-ups.

Correlation between cell free DNA levels and medical evaluation of disease progression in systemic lupus erythematosus patients

High levels of cell free DNA (cfDNA) in human blood plasma have been described in patients with autoimmune diseases. The aim of this study was to determine the levels of cfDNA in systemic lupus erythematosus (SLE) patients and to assess fluctuations of cfDNA concentrations compared to the course of disease progression under standard treatment. Therefore, nuclear cfDNA concentrations in plasma were measured in 59 SLE patients and 59 healthy controls. Follow-up blood plasma was collected from 27 of the 59 SLE patients. Patients were characterised by clinical parameters (antinuclear antibodies (ANA), anti-dsDNA-antibodies, C3, C4, and CRP), SLE disease activity index (SLEDAI) and medical therapy. Our results showed that cfDNA concentrations were significantly higher in SLE patients compared to healthy individuals. Levels of cfDNA assessed in serial samples correlated significantly with the medical evaluation of disease activity in SLE patients. Our results could implicate cfDNA as a global marker for disease activity.