Generating iPSCs with a High-Efficient, Non-Invasive Method-An Improved Way to Cultivate Keratinocytes from Plucked Hair for Reprogramming.

Various somatic cell types are suitable for induced pluripotency reprogramming, such as dermal fibroblasts, mesenchymal stem cells or hair keratinocytes. Harvesting primary epithelial keratinocytes from plucked human hair follicles (HFs) represents an easy and non-invasive alternative to a fibroblast culture from invasive skin biopsies. Nevertheless, to facilitate and simplify the process, which can be divided into three main steps (collecting, culturing and reprogramming), the whole procedure of generating hair keratinocytes has to be revised and upgraded continuously. In this study, we address advancements and approaches which improve the generation and handling of primary HF-derived keratinocytes tremendously, e.g., for iPSCs reprogramming. We not only evaluated different serum- and animal-origin-free media, but also supplements and coating solutions for an enhanced protocol. Here, we demonstrate the importance of speed and accuracy in the collecting step, as well as the choice of the right transportation medium. Our results lead to a more defined approach that further increases the reliability of downstream experiments and inter-laboratory reproducibility. These improvements will make it possible to obtain keratinocytes from plucked human hair for the generation of donor-specific iPSCs easier and more efficient than ever before, whilst preserving a non-invasive capability.

Opposite effect of negative and positive affect on stress procoagulant reactivity.

Exaggerated procoagulant responses to acute mental stress may contribute to coronary thrombosis, and continuing low-grade systemic coagulation activation may link negative affect with the development of coronary artery disease. We investigated whether negative and positive affect and perceived social support would moderate stress procoagulant reactivity. Psychological functioning, exhaustion, negative affectivity, depression, anxiety, worrying, vigor, and social support were assessed in 27 apparently healthy men (mean age 47 +/- 8 years) who underwent the 13-min Trier Social Stress Test combining preparation, speech, and mental arithmetic. Plasma levels of von Willebrand factor antigen (VWF:Ag), fibrinogen, factor VII clotting activity (FVII:C), FVIII:C, FXII:C, and D-dimer were measured immediately before and after stress. Acute stress elicited significant increases in hemodynamic, cortisol, and coagulant activity (p values < 0.05). VWF:Ag reactivity showed inverse relationships with exhaustion (r = -0.63, p < 0.001), negative affectivity (r = -0.53, p = 0.005), and worrying (r = -0.53, p = 0.005). Exhaustion and negative affectivity emerged as independent predictors of VWF:Ag reactivity explaining 54% of its variance. Fibrinogen reactivity showed inverse relationships with negative affectivity (r = -0.59, p = 0.002) and anxiety (r = -0.54, p = 0.005); negative affectivity emerged as an independent predictor of fibrinogen reactivity explaining 35% of its variance. Psychological functioning and FVII:C reactivity were also correlated (r = -0.52, p = 0.006). Whereas FVIII:C reactivity correlated positively with vigorous mood (r = 0.48, p = 0.012), positive associations between social support and procoagulant reactivity did not reach significance. Negative affect was associated with attenuated procoagulant reactivity to stress and the opposite was observed for positive affect. Negative affect is not likely to enhance the acute procoagulant stress response in healthy men.

Reduced glucocorticoid sensitivity of monocyte interleukin-6 production in male industrial employees who are vitally exhausted.

OBJECTIVE: Proinflammatory changes are thought to link vital exhaustion with adverse cardiovascular outcomes. Monocytes play a central role in the pathogenesis of atherosclerotic lesions and are a major source of circulating cytokines. We hypothesized that vital exhaustion may alter the regulation of monocyte activity, as measured by lipopolysaccharide (LPS)-stimulated and glucocorticoid inhibited release of the proinflammatory cytokine interleukin-6 (IL-6). METHODS: In 166 middle-aged apparently healthy men, vital exhaustion was measured by the Shortened Maastricht Exhaustion Questionnaire. Subjects in the highest quartile (highly exhausted, N= 38) were compared with those in the second and third quartiles (moderately exhausted N= 89) vs. those in the lowest quartile (nonexhausted, N= 39) in terms of plasma C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) levels, and as to IL-6 release after LPS stimulation in vitro. Inhibition of IL-6 release was determined by coincubation with increasing concentrations of dexamethasone. Monocyte glucocorticoid sensitivity was defined as the dexamethasone concentration inhibiting IL-6 release by 50%. RESULTS: Highly exhausted individuals had higher CRP levels than nonexhausted subjects (p=.008). LPS-stimulated IL-6 release was not significantly different between groups. However, in highly exhausted participants, dexamethasone was less able to inhibit IL-6 release (p=.010), and the glucocorticoid sensitivity was lower (p=.003) than in nonexhausted subjects. CONCLUSIONS: In highly exhausted individuals, glucocorticoids exert less suppressive action on monocyte IL-6 release than in nonexhausted subjects. This finding points to altered regulation of monocyte cytokine production as one possible pathway linking exhaustion with atherosclerosis.

Glucocorticoid sensitivity of circulating monocytes in essential hypertension.

BACKGROUND: Essential hypertension ranks among the strongest cardiovascular risk factors. Cytokine production by monocytes plays a key role in atherosclerosis development and acute coronary syndromes. We investigated whether stimulated monocyte cytokine release and its inhibition by glucocorticoids would differ between hypertensive and normotensive subjects. METHODS: Study participants were 222 middle-aged male employees with industrial jobs. Following the criteria of the World Health Organization/International Society for Hypertension, 76 subjects were classified as being hypertensive (systolic blood pressure > or = 140 mm Hg or diastolic blood pressure > or = 90 mm Hg). In vitro monocyte tumor necrosis factor (TNF)-alpha release after lipopolysaccharide (LPS) stimulation was assessed with and without coincubation with incremental doses of dexamethasone. Monocyte glucocorticoid sensitivity was defined as the dexamethasone concentration inhibiting TNF-alpha release by 50%. RESULTS: Hypertensive subjects showed 11% higher LPS-stimulated TNF-alpha release than normotensive subjects (F(1,181)= 5.21, P =.024). In hypertensive subjects, monocyte glucocorticoid sensitivity was 21% lower than in normotensive subjects (F(1,178)= 4.94, P =.027), indicating that dexamethasone inhibited relatively less TNF-alpha release in hypertensive subjects. Results held significance when a set of classic cardiovascular risk factors was controlled for. CONCLUSION: The findings suggest that proinflammatory activity of circulating monocytes is higher in hypertensive than in normotensive men, providing one potential pathway to explain the increased atherosclerotic risk with essential hypertension.

Allostatic load and work conditions.

Adverse work characteristics and poor social support have been associated with an increased risk for cardiovascular disease and other adverse health outcomes in otherwise apparently healthy adults. We undertook a cross-sectional study to evaluate the relationship between objective health status and work characteristics in industrial workers in Germany. Volunteers (n=324) were recruited from a representative random sample (n=537) of employees of an airplane manufacturing plant. Psychosocial work characteristics were assessed by the 52-item, 13-subscale salutogenetic subjective work analysis (SALSA) questionnaire, which assesses potentially salutogenic and pathogenic conditions. Factor analysis revealed three factors: decision latitude, job demands and social support. Biological health status was determined by the revised allostatic load score with 14 components: body-mass index, waist-to-hip ratio; systolic and diastolic blood pressure; plasma levels of C-reactive protein (CRP), tumor-necrosis factor-alpha, HDL, cholesterol, dehydroepiandrosterone sulfate; glycosylated hemoglobin; urinary cortisol, epinephrine, norepinephrine, and albumin. Score points were given for values in the high-risk quartile (maximum=14). General linear models revealed that older individuals and men had significantly higher allostatic load scores than younger participants or women. Of the SALSA factors, only job demands related significantly to allostatic load. The effect of demands was stronger in older individuals. Post-hoc analysis showed possible positive associations between high job demands and blood pressure or CRP, and between low social support and nocturnal excretion of cortisol or plasma levels of CRP. We conclude that this cross-sectional study on industrial employees found a weak association between a health summary score based on objective medical data and self-reported adverse work characteristics.

Contribution of the type 1 plasminogen activator inhibitor 4G/5G gene polymorphism to impaired fibrinolysis in vital exhaustion.

BACKGROUND: Inappropriate coping with chronic stress may result in a state of "vital exhaustion" that has been associated with coronary artery disease. Impaired fibrinolysis due to an increase in type 1 plasminogen activator inhibitor (PAI-1) might mediate this link. Genetic and environmental factors both regulate the plasma PAI-1 levels. We investigated the contribution of the PAI-1 4G/5G gene polymorphism to the plasma levels of PAI-1 in exhaustion. METHODS: The study participants were 258 (mean age 40.9 +/- 9.1 years) apparently healthy subjects of an airplane manufacturing plant in Germany who completed the Shortened 9-item Maastricht Vital Exhaustion Questionnaire. A median split was performed on exhaustion scores rendering two groups of exhausted and non-exhausted subjects. The PAI-1 4G/5G polymorphism in the promoter region of the PAI-1 gene and several variables related to the insulin resistance syndrome known to affect plasma PAI-1 levels were assessed. RESULTS: Across all subjects, exhausted individuals had higher PAI-1 antigen levels than non-exhausted subjects (46.6 +/- 20.7 vs 38.3 +/- 21.4 ng/ml, p = 0.002). There were no significant differences in the PAI-1 antigen levels between exhausted and non-exhausted individuals with both the 4G/4G and the 4G/5G polymorphism. With the 5G/5G polymorphism, however, exhausted subjects had higher PAI-1 antigen levels than non-exhausted subjects (44.9 +/- 22.9 vs 31.2 +/- 13.1 ng/ml, p = 0.017). These results did not change when controlling for the variables of insulin resistance. CONCLUSIONS: The findings suggest that the PAI-1 4G/5G gene polymorphism might affect the plasma PAI-1 levels related to exhaustion severity. With the 5G/5G polymorphism, exhausted subjects might have less fibrinolytic capacity than non-exhausted subjects.

Independent relation of vital exhaustion and inflammation to fibrinolysis in apparently healthy subjects.

OBJECTIVE: Vital exhaustion (VE) and a hypercoagulable state both have been associated with coronary artery disease (CAD). Candidate mechanisms by which VE predicts CAD events are impaired fibrinolysis and inflammatory changes, the latter also affecting hemostasis. We investigated whether VE and inflammation would independently relate to hemostasis. DESIGN: Study participants were 217 (mean age+/-SD, 40+/-9 years) apparently healthy men and women working at an airplane manufacturing plant in Germany who completed the Shortened 9-item VE Maastricht Questionnaire. All subjects had a set of classic cardiovascular risk factors assessed, and plasma levels of fibrin D-dimer, type I plasminogen activator inhibitor (PAI-1) antigen, C-reactive protein (CRP), and tumor necrosis factor (TNF)-alpha were measured. RESULTS: PAI-1 correlated with VE (r=0.18, p=0.009), CRP (r=0.20, p=0.004), and TNF-alpha (r=0.18, p=0.009); D-dimer correlated with CRP (r=0.16, p=0.018). In linear regression analyses, VE and TNF-alpha independently explained 2 and 1%, respectively, of the variance in PAI-1. CONCLUSION: Our study corroborates previous findings on impaired fibrinolysis in VE. The findings suggest that VE and inflammation may impair fibrinolysis by different pathways, and independently of traditional cardiovascular risk factors.

Relationship between overnight neuroendocrine activity and morning haemostasis in working men.

Sustained effects of SNS (sympathetic nervous system) and HPAA (hypothalamic-pituitary-adrenal axis) hyperactivity on haemostasis have not been investigated. In the present study, we tested for an association of overnight urinary catecholamine and cortisol excretion with morning plasma levels of fibrinogen, PAI-1 (plasminogen activator inhibitor-1) and D-dimer. Participants (639 male industrial employees) with a complete dataset were studied (age, 41+/-11 years; mean+/-S.D.). Subjects collected overnight urinary samples and had a fasting morning blood sample drawn. Measurement of urinary adrenaline (epinephrine), noradrenaline (norepinephrine) and cortisol were dichotomized to perform multivariate analyses of (co)variance. Haemostatic parameters were measured by ELISA. Fibrinogen was higher in men with high adrenaline (F(7,631)=5.68, P=0.018; where the subscripted value represents the degrees of freedom) and high noradrenaline (F(7,631)=4.19, P=0.041) compared with men with low excretion of the respective hormones. PAI-1 was higher in men with high cortisol than in men with low cortisol (F(7,631)=4.77, P=0.029). Interaction revealed that subjects with high cortisol/low noradrenaline had higher PAI-1 than subjects with low cortisol/high noradrenaline (P=0.038). Subjects with high adrenaline/high noradrenaline had higher D-dimer than subjects with high adrenaline/low noradrenaline (P=0.029), low adrenaline/high noradrenaline (P=0.022) and low adrenaline/low noradrenaline (not significant). When covariance for several confounders of haemostatic function was determined, the main effect of adrenaline on fibrinogen and the interaction between adrenaline and noradrenaline for D-dimer maintained significance. Although overnight SNS hyperactivity was associated independently with morning hypercoagulability, the relationship between the activity of HPAA and haemostasis was mediated by traditional cardiovascular risk factors.

Assessment of exhaustion and related risk factors in employees in the manufacturing industry--a cross-sectional study.

OBJECTIVES: Vital exhaustion, a construct overlapping with burnout, is an independent risk factor for adverse health outcomes, including cardiovascular disease. We aimed to assess vital exhaustion in employees in the manufacturing industry and identify work characteristics associated with exhaustion. METHODS: Cross-sectional study. A stratified, representative random sample of employees from a manufacturing plant for airplane parts and assemblies was invited ( n=647), of whom 537 employees (83% accrual) volunteered to participate. Scores obtained by the nine-item Shortened Maastricht Exhaustion Questionnaire were compared with the mental and physical summary scales of the SF-12 General Health Survey and to a list of 20 health complaints. Pathogenic and salutogenic work characteristics were assessed by the self-reported 52-item, 13-subscale SALSA questionnaire. RESULTS: Vital exhaustion correlated with the mental summary score of the SF-12 and the number of health complaints and was positively associated with age. Exhaustion was not associated with gender, position (socio-economic status) or being on a wage (paid per completed item up to a contracted amount) or salary (payment as fixed monthly income). The instrument identified departments with high levels of exhaustion. Of the observed variance in exhaustion, 29% was explained by age, department, and five work characteristics: High levels of exhaustion (score >10) were related to excessive workload or qualitative demands (scoring in the highest quartile; OR(adj) 7.5; 95% CI 2.4-23), to adverse physical work conditions (OR(adj) 6.9; 95% CI 2.2-21), to adverse co-worker behavior (OR(adj) 4.8; 95% CI 1.4-16), to qualification potential (OR(adj) 0.32; 95% CI 0.11-0.97), and to social support by co-workers (OR(adj) 0.34; 95% CI 0.13-0.99), respectively. CONCLUSIONS: The nine-item instrument allows rapid screening of employees for self-reported levels of exhaustion. Besides physical work conditions and workload, absence or presence of social support by co-workers is strongly associated with exhaustion.