The Covert Surge: Murine Bile Acid Levels Are Associated With Pruritus in Pediatric Autoimmune Sclerosing Cholangitis.

Objectives: The exact etiology of pruritus in chronic cholestasis is unknown. Pruritus intensity does not correlate with common biochemical indices and there is a lack of biomarkers guiding diagnosis and treatment. We explored profiles of bile acids (BA) and muricholic acids (MCA) as well as autotaxin (ATX) antigen levels as potential circulating biomarkers of pruritus in pediatric patients. Methods: In 27 pediatric cholestatic patients [autoimmune sclerosing cholangitis (ASC) n = 20 (with pruritus n = 6, without pruritus n = 14); progressive familial intrahepatic cholestasis (PFIC) n = 7 (with pruritus n = 5, without pruritus n = 2)] and 23 age-matched controls pruritus was assessed by a visual analog scale of pruritus (PVAS). We obtained profiles of serum human BA including MCA using a mass-spectrometry assay and ATX antigen levels with a commercial ELISA. Results: PFIC and ASC patients exhibited significantly higher BA-, and MCA levels, than healthy controls, but only PFIC patients showed elevated ATX antigen levels higher [median: 1,650 ng/ml, interquartile rang (IQR): 776.9-3,742] compared to controls (median: 315.9 ng/ml, IQR: 251.1-417.2; PFIC p = 0.0003). ASC patients with pruritus showed only a minor increase in total BA (tBA) levels (median: 76.5 µmol/L, IQR: 54.7-205), but strikingly higher T-conjugated BA (median: 16.4 µmol/L, IQR: 8.9-41.4) and total MCA (tMCA) (median: 1.15 µmol/L, IQR: 0.77-2.44) levels compared to ASC patients without pruritus (tBA median: 24.3 µmol/L, IQR: 16.2-80.8; p < 0.0408; T-conjugated BA median: 1.3 µmol/L, IQR: 0.8-4.9; p = 0.0023; tMCA median: 0.30 µmol/L, IQR: 0.13-0.64, p = 0.0033). BA/MCA profiles distinctly differed depending on presence/absence of pruritus. Different from PFIC patients, ATX antigen levels were not significantly elevated in ASC patients with (median: 665.8 ng/ml, IQR: 357.8-1,203) and without pruritus (median: 391.0 ng/ml, IQR: 283.2-485.6). In ASC patients, tBA, tMCA, and ATX antigen levels did not correlate with pruritus severity. Conclusion: Despite the same underlying disease, pediatric ASC patients with pruritus exhibit significantly altered BA profiles and MCA levels compared to ASC patients without pruritus. ATX antigen levels seem to have little diagnostic or prognostic meaning in ASC patients. An increased ATX activity alone seems not to be causal for pruritus genesis in ASC patients. Clinical Trial Registration: [www.drks.de], identifier [DRKS00026913].

Bile acid-induced tissue factor activity in hepatocytes correlates with activation of farnesoid X receptor.

Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.

Self-referential processing and perspective taking in patients with a borderline personality disorder.

Divergent self- and other-referential processes play a particular role in the development and maintenance of borderline personality disorder (BPD). This study investigated self-referential processes in patients with BPD and age-matched controls. Participants performed a trait-judgment task, taking their own and the perspective of a close other person. Memory was assessed during recall of the previous choices. Results revealed over all more negative self-appraisals in patients than controls, which seemed due to making less positive self-referential choices rather than an increased choice of negative traits. Interestingly, taking another perspective, patients had a healthier, predominantly positive self-assessment, albeit still attenuated compared to controls. The characteristics of the appraisals were mirrored in memory performances. Moreover, self-esteem seems to be a potential protective factor, as self-appraisals were more positive with higher self-esteem. Altogether, this study shows significantly deviant self-referential processes in patients with BPD, suggesting that patients do not integrate what they believe others think about them into their self-concept.

Impact of electric cardioversion on platelet activation.

INTRODUCTION: Atrial fibrillation (AF) comes along with high risk of stroke. This risk continues even after re-establishing sinus rhythm with cardioversion. Aim of this study is to evaluate the contribution of electric cardioversion (EC) to platelet activation and procoagulatory tendency. METHODS: Extent of platelet activation before and after electric cardioversion was quantified using flow cytometry, impedance aggregation measurements with Multiplate®, and quantification of serum levels of platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) in patients with AF (N = 10). RESULTS: No significant differences were observed in any of the measured parameters comparing the values from before and after cardioversion. Geometric means of P-selectin expression and integrin αIIbß3 activation were 0.27 (+/- 0.07) and 2.30 (+/- 2.61) before EC and 0.28 (+/- 0.17) and 1.67 (+/- 1.82) after EC. Levels of ß-TG were 110.11 ng/ml (+/- 3.78) before and 110.51 ng/ml (+/- 2.56) after EC, levels of PF4 were 35.64 ng/ml (+/- 12.94) before and 32.40 ng/ml (+/- 4.95) after EC. Platelet aggregation triggered with adenosine diphosphate (ADP), arachidonic acid, collagen, Ristocetin, or thrombin receptor activating peptide (TRAP) revealed results within the normally expected ranges without significant changes before and after EC. DISCUSSION: Electric cardioversion has no influence on platelet activation markers which is in agreement with other studies reporting electrical cardioversion to be safe.

Borderline personality disorder and childhood trauma: Exploring the buffering role of self-compassion and self-esteem.

OBJECTIVES: The aim of the present study was to investigate whether patients with borderline personality disorder (BPD) show lower self-compassion and self-esteem than healthy controls and whether patients' self-compassion and self-esteem moderate the association between childhood trauma and the severity of their BPD symptoms. METHOD: Self-reported self-compassion, self-esteem, and the current severity of BPD symptoms were assessed in 35 female patients with BPD and 35 age-matched control participants. Further, traumatic childhood experiences were recorded in the patient group. RESULTS: Patients with BPD reported significantly lower self-compassion and self-esteem compared to healthy controls. In addition, self-compassion but not self-esteem moderated the positive correlation between childhood trauma and the severity of BPD symptoms. DISCUSSION: Self-compassion appears to buffer the negative consequences of childhood traumatization. Therefore, cultivating self-compassion may be an important therapeutic aim for patients with BPD.

[Supporting Interventions for Families with Children of Mentally Ill Parents: An Overview of Family-Oriented Complex Interventions in German-Speaking Countries]. / Familien mit Kindern psychisch kranker Eltern: Ein Überblick über ambulante, familienorientierte Komplexangebote im deutschsprachigen Raum.

OBJECTIVE: The aim of the present review was to identify all outpatient, family-oriented complex interventions for children of mentally ill parents known in the German-speaking countries on the basis of defined minimum requirements and to give an overview of their structure, content and proof of effectiveness. METHODOLOGY: The interventions were identified by means of internet and literature research. If the defined criteria were met, the providers were contacted and asked to participate in a written survey. RESULTS: A total of 512 offers could be identified, 46 of which were to be classified as family-oriented complex interventions. Only a few interventions have been systematically evaluated so far. CONCLUSION: There is a large number of interventions for the children of mentally ill parents, but family-oriented complex interventions are rare. There is also a considerable need for evaluation studies.

Increased tissue factor activity promotes thrombin generation at type 1 diabetes onset in children.

OBJECTIVE: In type 1 diabetes (T1D), a prothrombotic status due to elevated coagulation factors coincides with metabolic derailment. In a previous study, we discovered altered thrombin generation profiles in children with T1D. These alterations are potentially most pronounced at T1D onset and ameliorated after insulin treatment. We tested this hypothesis in a longitudinal study, measuring thrombin generation together with coagulation parameters in children at T1D onset and during follow-up. MATERIALS AND METHODS: Twenty-three children (12 female, age: 9.4 [2.7-17.3] years; median [range]) were tested at T1D onset and after long-term insulin treatment. Thrombin generation was measured using calibrated automated thrombography. Tissue factor (TF) activity and tissue factor pathway inhibitor (TFPI) activity were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: A procoagulant shift was observed in thrombin generation traces at T1D onset compared to follow-up (time to peak: 5.67 [4.11-7.67] min vs 6.39 [4.89-10.44] min, P < .001). These alterations at T1D onset coincided with increased TF activity (5.18 [0.01-12.97] pmol/L vs 2.67 [0.04-10.41] pmol/L, P < .05) and increased TFPI activity (0.051 [0.038-0.074] U/mL vs 0.035 [0.026-0.056] U/mL, P < .05). CONCLUSION: The procoagulant shift in thrombin generation at T1D onset is a result of increased TF activity, but this effect is partially counterbalanced by increased TFPI levels. Elevated TF and TFPI levels hint to a fragile hemostatic balance at the endothelial lining of blood vessels. Additional prothrombotic stimuli may tip over this balance explaining the increased thrombotic risk of children with T1D.

New insights into neonatal coagulation: normal clot formation despite lower intra-clot thrombin levels.

BACKGROUND: Healthy neonates exhibit no bleeding tendencies, but exhibit longer partial thromboplastin times than adults. Lower clotting factor levels may be balanced by lower inhibitor levels, which is not reflected in routine coagulation assays, but could result in normal clot formation in vivo. The novel thrombodynamics assay simulates a damaged vessel with tissue factor immobilized to a surface. We hypothesized that intra-clot thrombin levels and spatial fibrin clot formation with this assay are comparable in neonates and adults. METHODS: Coagulation was tested in plasma from venous neonatal blood (N = 12), cord blood (N = 30), and adult blood (N = 20) using thrombodynamics and calibrated automated thrombography. RESULTS: Neonates exhibited a higher initial rate of clot formation than adults (adult: 60.7 ± 3.9 µm/min; neonatal: 66.8 ± 3.9 µm/min; cord: 68.1 ± 3.3 µm/min; P < 0.001) and a comparable stationary rate of clot formation (adult: 35.8 ± 8.5 µm/min; neonatal: 37.0 ± 4.6 µm/min; cord: 36.0 ± 5.2 µm/min; P = 0.834). Intra-clot thrombin levels were lower in neonates (adult: 41.9 ± 11.2 AU/l; neonatal: 22.6 ± 10.2 AU/l; cord: 23.6 ± 9.7 AU/l; P < 0.001), but the longitudinal rate of thrombin propagation was comparable (adult: 27.2 ± 4.2 µm/min neonatal; 27.9 ± 2.9 µm/min; cord: 27.6 ± 3.4 µm/min; P = 0.862). CONCLUSIONS: Despite lower intra-clot thrombin levels, neonates exhibit normal spatial fibrin clot growth, which concurs with clinically well-functioning hemostasis in healthy neonates.

Neonatal thrombocytopenia: Thrombin generation in presence of reduced platelet counts and effects of rFVIIa in cord blood.

Healthy neonates exhibit a well-functioning haemostatic system despite peculiarities regarding composition of clotting factors and inhibitors as well as impaired platelet aggregation. Thrombocytopenia and severe bleeding events are feared in sick infants. Recombinant factor VIIa (rFVIIa) is a haemostatic agent used as a last resort in neonates with refractory bleedings. Aim of this study was to investigate in-vitro (i) changes in thrombin generation with different platelet counts, (ii) effects of rFVIIa under conditions of thrombocytopenia and (iii) potentially differing dose-response of rFVIIa in cord blood as a surrogate for neonatal blood compared to adult blood. Thrombin generation parameters were observed in cord blood plasma and adult plasma with various platelet counts, with or without addition of rFVIIa, respectively. Low platelet counts did not influence thrombin generation in cord blood in contrast to adult blood. RFVIIa primarily affected lag time throughout all platelet concentrations. Interestingly, peak height was reduced exclusively in cord blood plasma after addition of rFVIIa. No significant differences regarding dose-response were observed between cord blood and adult blood. In contrast to adult blood, thrombocytopenia in cord blood does not significantly influence thrombin generation. Even at very low platelet counts there is enough negatively charged surface to support rFVIIa action in plasma from cord blood and adult blood in-vitro.

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels.

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities. Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood. Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208-0.026 µg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography. Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2-71.8%] of vehicle; cord: 73.4% [IQR: 70.2-91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 µg/ml; cord: 0.814 µg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels. Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates.

Non-enzymatic quantification of polyphosphate levels in platelet lysates and releasates.

Inorganic polyphosphate has been shown to be shed upon platelet activation inducing prothrombotic stimuli on the coagulation system. Several methods have been published to detect and quantify polyphosphate in various cells and tissues, but evaluation of platelet content has only been achieved by indirect detection of orthophosphate after enzymatic digestion, thus, relying heavily on specificity of an exopolyphosphatase that is not commercially available. We present a non-enzymatic method for quantification of platelet-derived polyphosphate featuring optimized extraction on silica spin-columns, followed by specific fluorescence detection using DAPI. This allowed us to quantify polyphosphate in platelet lysates, but also in releasates of TRAP-activated platelets for the first time. Extraction of exogenous polyphosphate from buffer and sample matrices resulted in quantitative yields while removing matrix effects observed with direct fluorescence detection. Treatment of eluted fractions with phosphatase completely abrogated polyphosphate-specific fluorescence arguing for no additional compounds influencing the fluorescence detection. This was confirmed by no change in fluorescence intensity in samples previously treated with DNase and RNase. Taken together, we developed a robust and easily standardizable method to quantify polyphosphate in platelet lysates and releasates that will facilitate polyphosphate related investigations of platelet physiology and coagulation.